WO2023172495A2 - Methods and compositions for production of biological substances in a genetically modified cell - Google Patents

Abstract

The present invention provides genetically modified cells for the production of biological substances, said cells comprising a first polynucleotide encoding a fusion polypeptide comprising global metabolic regulator, or a component or functional fragment or derivative thereof, operably linked to a signal peptide enabling the secretion of the fusion polypeptide, and optionally one or more additional polynucleotide(s) encoding at least one polypeptide encoding the biological substance or a polypeptide that participates in the production of a biological substance. Also provided are various nucleic acids and expression constructs encoding the fusion polypeptides and/or biological substances, which may comprise any of various promoters, as well as methods of making and using the genetically modified cells.

Description

METHODS AND COMPOSITIONS FOR PRODUCTION OF BIOLOGICAL SUBSTANCES IN A GENETICALLY MODIFIED CELL

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63/317,403, filed March 7, 2022, the disclosure of which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention provides genetically modified cells expressing a fusion polypeptide comprising a global metabolic regulator, or a component or functional fragment or derivative thereof, operably linked to a signal peptide, thereby enabling the secretion of the fusion polypeptide, e.g., to a space outside of the cytoplasmic membrane of the cell. The cells may optionally comprise one or more additional polynucleotides encoding additional polypeptide(s), which may be a biological substance, or participate in the production of a biological substance. Also provided are various nucleic acids and expression constructs encoding the fusion polypeptides and/or biological substances, which may comprise any of various promoters, as well as methods of making and using the genetically modified cells.

BACKGROUND

[0003] Microbial cells are equipped with a complex sensory system allowing them to sense changes happening intracellularly and in their environments. The detected signals are transmitted into the transcriptional regulatory systems, which eventually results in a response at the transcriptional and post-transcriptional levels. This response enables cells to adapt appropriately to the changes and facilitate further proliferation, or switch to a survival mode (Janga et al., 2007). A major aspect of these functional responses comprises regulation of cellular metabolism (Gama-Castro et al., 2008).

[0004] At the transcriptional level, metabolic regulation is brought about by a complex network of transcription factors (TFs). Most TFs are proteins with a DNA-binding domain and an allosteric site that may bind metabolites either non-covalently, and/or undergo covalent modification by enzymes to modulate the regulatory activity of the TF (Seshasayee et al., 2006). In E. coh. about 8%, or roughly 300 genes are predicted or known TFs (Perez-Rueda and Collado- Vides, 2000). The TFs are arranged into a network of interacting cascades with the ones in the top tier of the hierarchy participating directly or indirectly in regulation of a large number of other TFs and, correspondingly, in regulation of operons that belong to different metabolic pathways. These TFs are considered global regulators. It has been shown that only seven regulatory proteins (CRP, FNR, IHF, FIS, ArcA, NarL, and Lrp) modulate the expression of 51% of genes in E. coli. NarL, Fur, Mlc, CspA, Rob, PurR, PhoB, CpxR, SoxR/SoxS, OxyR, PdhR, ModE, FlhA, CysB, DnaA, BolA, IciA are positioned in a second level of this hierarchy because they also initiate regulatory cascades (Martinez-Antonio and Collado- Vides, 2003).

[0005] The other class of global regulators acting at the level of transcription is sigma factors. Sigma factors reversibly bind the core subunit of RNA polymerase (RNAP) to endow promoter specificity on the polymerase holoenzyme, thus mediating transcription of all genes in bacteria. [0006] There are seven sigma factors in A. coli. Expression of alternative sigma factors provides a powerful mechanism to control the expression of discrete sets of genes in response to specific nutritional, developmental, or stress-related signals (Heimann, 2019).

[0007] In contrast to the global regulatory effect manifested at the level of transcription, most post-transcriptional regulatory mechanisms are specific for individual targets. However, such proteins as Hfq, CsrA (Nogueira and Springer, 2000), Obg (Starosta et al., 2014) and toxinantitoxin (TA) modules (Harms et al., 2018; Sauert et al., 2016), which exert their regulatory functions at the post-transcriptional level, can be clearly attributed to the network of global regulators.

[0008] Toxin-antitoxin (TA) modules are widespread in prokaryotic genomes (Gerdes et al. 2005; Harms et al., 2018; Horak and Tamman 2017; Van Melderen 2010; Yamaguchi and Inouye, 2011). The E. coli K-12 MG1655 strain has at least 37 TA loci. The toxins expressed from TA loci use a wide variety of molecular activities to interfere with such cellular functions as replication, translation, and cell wall synthesis to inhibit bacterial growth. Such toxins are able to cleave, degrade, or modify their cellular targets enzymatically, and thus can obstruct bacterial physiology even at low protein concentrations. Antitoxins are proteins or RNAs that control their cognate toxins through direct interactions, and through transcriptional and translational regulation of TA module expression. Accordingly, TA modules are categorized into six different types, depending on how the antitoxin neutralizes expression and/or activity of the toxin (Harms et al. 2018). The major biological functions of TA modules are post- segregational killing (Gerdes et al., 1986), abortive infection (Dy et al., 2014), and persister formation/antibiotic tolerance (Harms et al., 2016).

[0009] The E. coli toxin-antitoxin (TA) module MazEF was identified as a post- transcriptional metabolic regulator which globally affects protein synthesis in response to a variety of different stress conditions (Sauert et al., 2016; Vesperet al., 2011; Zhang et al., 2003). It consists of a labile antitoxin MazE and a stable toxin MazF, which is a sequence-specific endoribonuclease that preferentially cleaves messenger RNAs (mRNAs) at the 3’ end of the first A base in an ACA sequence in a ribosome-independent manner (Zhang et al., 2003b, 2005; US Patent Nos. 8,183,011; 9,243,234). These proteins are encoded by the genes mazE and mazF organized in an operon located downstream of the relA gene (Aizenman et al., 1996). [0010] During exponential growth, one MazE dimer forms a stable TA complex with two MazF dimers and neutralizes the toxin. Besides, this complex along with the MazE protein serves as a repressor for the mazEF operon (Zhang et al., 2003a). Therefore, expression of the operon is strongly repressed (Marianovski et al. 2001) and the toxic effect of the mRNA interferase is not exerted under these conditions (Engelberg-Kulka et al., 2004; Gerdes et al., 2005). However, any environmental stress causing growth inhibition leads to the degradation of the MazE antitoxin by ATP-dependent serine proteases ClpAP and Lon (Aizenman et al., 1996; Christensen et al., 2003) and release of the unbound MazF toxin in the cell. The released MazF attacks and cleaves mRNAs, thus inhibiting protein synthesis and the cellular growth (Inouye, 2006). These adverse environmental conditions promote de-repression of the mazEF operon as well (Muthuramalingam et al. 2016).

[0011] The multilevel regulatory system managing cellular metabolism is closely intertwined with complex signaling networks responsible for sensing environmental and intracellular conditions and the status of the cell envelope and cytoplasmic membrane, as well as for transmission of the registered signals. This surveillance involves several classes of sensor proteins: histidine kinases; chemoreceptors; membrane components of the sugar phosphotransferase system; adenylate, diadenylate and diguanylate cyclases and certain cAMP, c-di-AMP and c-di-GMP phosphodiesterases; extracytoplasmic function sigma factors; and, Ser/Thr/Tyr protein kinases and phosphoprotein phosphatases (Galperin 2018).

[0012] The complex network of interactions in the cellular sensing/regulatory pathways has developed to secure the long-term survival and proliferation of a microbial population while competing with other species for limited resources in their natural habitats. Since the environmental conditions are constantly changing, sometimes abruptly, the viability and competitiveness of microorganisms depend on the capacity for tight regulation of gene expression and post-translational modifications to ensure that adequate amounts of the right proteins are timely produced in response to specific internal and external stimuli (Gottesman, 2019; Lee and Kim, 2015; Seshasayee et al., 2006).

[0013] On the contrary, development of overproducing strains, which is the basis of the modem industrial microbial production, necessarily includes a great deal of artificial modifications in the genetic patterns of microorganisms resulting in dysregulation of the natural sensing/regulatory pathways. These modifications aim to remove genetic obstacles in rerouting metabolic fluxes towards production of the desired biological substances on one hand, and improving the physiological performance of the altered microorganisms on the other (Fiedurek et al., 2017; Lee and Kim, 2015; Sanchez and Demain, 2008; Tamano, 2014).

[0014] Most metabolic pathways are not limited by a single rate-limiting step and optimized pathways require a balanced expression of several enzymes. Without such coordination, metabolic imbalance can lead to the accumulation of gene products or intermediate metabolites with potentially cytotoxic effects or, in some cases, may result in depletion of a metabolite needed for normal cell growth (Koffas et al., 2003; Pitera et al., 2007). Furthermore, the overexpression of genes/proteins often results in undue metabolic burden on the cell (Glick, 1995).

[0015] Since the success of strain development is defined by the ability to achieve an optimal balance between altered pathway expression and cell viability, several metabolic engineering techniques that can affect the cell in a global fashion, such as artificial transcription factor engineering, global transcription machinery engineering, ribosome engineering, and genome shuffling, have been developed (Santos and Stephanopoulos, 2008).

[0016] Global transcription machinery engineering (gTME) is a powerful tool that enables a complete reprogramming of the cellular transcriptome. It does so through the targeted mutagenesis, e.g., via error-prone PCR, of select components of the transcriptional machinery, particularly those involved in DNA sequence recognition and thus dictate the promoter preferences of RNA polymerase (Alpert et al. 2006; Santos and Stephanopoulos, 2008). In prokaryotic systems, sigma factors bind to the promoter regions of genes with varying degrees of affinity and help to preferentially recruit the RNA polymerase holoenzyme to initiate transcription. Thus, slight variations in these proteins have the potential to greatly affect the subset of genes that are bound by RNA polymerase and expressed. The introduction of plasmid- encoded mutants of the principal sigma factor in E. coll (c⁷⁰) resulted in strains optimized for a variety of phenotypes, including ethanol tolerance, lycopene overproduction, and simultaneous tolerance to sodium dodecyl sulfate and ethanol (Alper and Stephanopoulos, 2007; U.S. Patent No. 9,273,307). Genetic manipulations with sigma factors aiming at microbial strain improvement are also described in other US patents (U.S. Patent Nos. 5,200,341; 5,686,283; 6,156,532; 10,760,108).

[0017] Industrial microorganisms redesigned for overproduction of target substances in large-scale production fermentation processes encounter simultaneously various environmental challenges, which do not usually exist in their natural habitats. Among these challenges are stresses originated from poor mixing (increased mixing time and formation of dead zones in the production scale fermenters), which result in gradients in concentration of nutrients, pH, temperature, dissolved oxygen, dissolved CO2 (Bylund et al, 1998; Enfors et al., 2001; Lara et al., 2006; Lee, 1996; Singh et al., 1986; Vrabel et al., 1999) and increased formation of detrimental by-products (Bylund et al., 2000; Castan and Enfors, 2001; Xu et al., 1999). High hydraulic pressure, surplus of heat generated by agitation and metabolic processes, high viscosity of culture broth, toxic impurities contained in industrial-grade raw materials may also exert negative effects on physiology of the microorganisms (Schmidt, 2005; Takors, 2012; Wehr et al., 2019).

[0018] Development of overproducing strains necessitates rerouting metabolic fluxes towards production of the desired substances on one hand, and improving the physiological performance of the altered microorganisms on the other (Fiedurek et al., 2017; Lee and Kim, 2015; Sanchez and Demain, 2008; Tamano, 2014). However, metabolic imbalance can lead to the accumulation of gene products or intermediate metabolites with potentially cytotoxic effects or, in some cases, may result in depletion of a metabolite needed for normal cell growth (Koffas et al., 2003; Pitera et al., 2007). Furthermore, the overexpression of genes/proteins often results in undue metabolic burden on the producing cell (Glick, 1995).

[0019] The general metabolic strategy all microorganisms use to survive and proliferate in nature is based on the maximal uptake of available nutrients from the environment, most often at the expense of efficiency of utilization. This intrinsic property of microorganisms causes significant problems when they are transferred into artificial, nutrient-rich conditions of research or industrial fermentations. The imbalanced fast growth in these artificial environments is usually associated with growth yields lower than 50% of the utilized substrate, accumulation of high concentrations of by-products, some of which are toxic, high requirements for oxygen and high rates of heat generation. All of these factors may significantly affect efficiency and cost-effectiveness of industrial fermentations (Lee, 1996). Currently, the rate of substrate uptake and, correspondingly, the specific growth rate of microbial cultures in artificial conditions is regulated by external factors: either by composition of nutrient medium and the use of various feeding strategies limiting cellular growth (growth limitation by substrate availability), or by changing physico-chemical conditions (for instance, lowering temperature). However, these are suboptimal methods. Implication of all the feeding techniques in large scale industrial fermentations is accompanied by appearance of gradients in substrate and by-product concentrations through the volume of fermentor as well as in oxygen availability (Schmidt, 2005; Takors, 2012; Wehr et al., 2019). Nutrient limitation, though broadly used in the fermentation industry (Choi et al., 2006; Lee, 1996; Riesenberg and Guthke, 1999; Shiloach and Fass, 2005), induces both the stringent response and the general stress response in the cell (Shiloach and Fass, 2005; Teich et al., 1999); besides product synthesis may be limited by carbon and energy sources under these conditions (Sanden et al., 2003). Cultivation at low temperatures is not always technically feasible in the industry since large-scale fermentors usually are operated at maximum cooling capacity (Hensing et al., 1995). Low temperature is also known as one of the typical environmental stress factors for microorganisms (Mattanovich et al., 2004).

SUMMARY OF THE INVENTION

[0020] As specified in the Background section above, there is a great need in the art for genetically modified cells, e.g., microbial cells, and methods for the scalable production of biological substances. The present application addresses these and other needs.

[0021] Disclosed herein are genetically modified cells expressing a fusion polypeptide comprising a global metabolic regulator, or component or functional fragment or derivative thereof, operably linked to a signal peptide, thereby enabling the secretion of the fusion polypeptide, e.g., to a space outside of the cytoplasmic membrane of the cell. The cells may optionally comprise one or more additional polynucleotides encoding additional polypeptide(s), which may be a biological substance, or which may participate in the production of a biological substance. Further disclosed herein are various nucleic acids and expression constructs encoding the fusion polypeptides and/or biological substances, which may comprise any of various promoters, as well as methods of making and using the genetically modified cells.

[0022] In one aspect is provided a genetically modified cell, said cell comprising a first polynucleotide encoding a fusion polypeptide comprising a global metabolic regulator, or a component or functional fragment or functional fragment or derivative thereof, operably linked to a signal peptide which enables secretion of the fusion polypeptide to the space outside of the cytoplasmic membrane of said cell.

[0023] In some embodiments, the expression of the fusion polypeptide in said cell does not fully inhibit growth of said cell.

[0024] In some embodiments, the growth rate of said cell during the expression of the fusion polypeptide is higher than 0. [0025] In some embodiments, the global metabolic regulator operably linked to the signal peptide is a toxin component of a toxin and antitoxin (TA) module or a functional fragment or functional fragment or derivative thereof.

[0026] In some embodiments, the fusion polypeptide comprises the same toxin component as the toxin component of an endogenous TA module of said cell, and the activity of said endogenous TA module of said cell is eliminated.

[0027] In some embodiments, the genetically modified cell described herein may further comprise one or more additional polynucleotides, said additional polynucleotide(s) encoding at least one polypeptide which is a biological substance or which participates in the production of the biological substance.

[0028] In some embodiments, the first polynucleotide and/or the one or more additional polynucleotide(s) is modified to replace one or more nucleotide sequences in a corresponding mRNA(s) that are recognizable by said toxin component, or the functional fragment or functional fragment or derivative thereof, so that such mRNA(s) becomes resistant to the destruction by said toxin component.

[0029] In some embodiments, the cell is a microbial cell.

[0030] In some embodiments, the microbial cell is a prokaryotic cell.

[0031] In some embodiments, the prokaryotic cell is a bacterial cell.

[0032] In some embodiments, the prokaryotic cell is from a genus selected from Nocardia, Acelobacler, Bacillus, Clostridium, Corynebacterium, Erwinia, Escherichia, Gluconacetobacter , Gluconobacter , Klebsiella, Lactobacillus, Lactococcus, Mannheimia, Propionibacterium, Pseudomonas, Ralstonia, Saccharopolyspora, Salmonella, Serratia, Streptomyces, Xanthomonas, and Zymomonas.

[0033] In some embodiments, the prokaryotic cell is from a species selected from Acetobacter aceti, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus subtilis, Clostridium acetobutylicum, Clostridium butyricum, Clostridium thermoaceticum, Clostridium tyrobutyricum, Corynebacterium glutamicum, Erwinia citreus, Erwinia herbicola, Gluconacetobacter hansenii, Gluconobacter oxydans, Klebsiella oxytoca, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Mannheimia succinicip-roducens, Nocardia lactamdurans, Propionibacterium shermanii, Pseudomonas denitrificans, Ralstonia eutropha, Saccharopolyspora erythrea, Saccharopolyspora spinosa, Serratia marcescens, Streptomyces clavuligerus, Streptomyces griseus, Streptomyces lividans, Streptomyces roseosporus, Xanthomonas campestris, Zymomonas mobilis, Escherichia coli, Lactococcus lactis, Bacillus cereus, Salmonella typhimurium, and Pseudomonas fluor escens. [0034] In some embodiments, the prokaryotic cell is from a species selected from Escherichia coh, Lactococcus lactis, Bacillus subliHs. Bacillus cercus, Salmonella lyphi murium, and Pseudomonas fluor escens.

[0035] In some embodiments, the prokaryotic cell is from the species Escherichia coli.

[0036] In some embodiments, the signal peptide enables secretion of the fusion polypeptide to the periplasm of the microbial cell.

[0037] In some embodiments, the signal peptide enables secretion of the fusion polypeptide to the extracellular space of the microbial cell.

[0038] In some embodiments, the cell is a eukaryotic cell.

[0039] In some embodiments, the eukaryotic cell is a fungal cell.

[0040] In some embodiments, the eukaryotic cell is from a genus selected from Chrysosporium, Eremothecium (Ashbya), Rhizopus, Acremonium (Cephalosporium), Aspergillus, Arxula, Blakeslea, Candida, Fusarium, Ganoderma, Hansenula, Kluyveromyces, Mortierella, Mucor, Pachisolen, Penicillium, Phaffia, Pichia, Saccharomyces, Schizosaccharomyces, Trichoderma, Umbelopsis, Yarrowia, and Zy go saccharomyces.

[0041] In some embodiments, the eukaryotic cell is from a species selected from Acremonium chrysogenum, Arxula adeninivorans, Aspergillus awamori, Aspergillus chrysogenum, Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, Candida boidinii, Candida sorenensis, Blakeslea trispora, Chrysosporium lucknowense, Eremothecium (Ashbya) gossypii, Fusarium venenatum, Ganoderma lucidum, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Mortierella alpine, Mucor miehei, Pachysolen tannophilus, Penicillium brevicompactum, Penicillium chrysogenum, Phaffia rhodozyma, Pichia methanolica, Pichia stipitis, Rhizopus oryzae, Trichoderma reesei, Umbelopsis isabellina, Yarrowia lipolytica, Zygosaccharomyces bailii, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe.

[0042] In some embodiments, the eukaryotic cell is from a species selected from Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe.

[0043] In some embodiments, the signal peptide is a Sec-dependent signal peptide or a twin- arginine translocation (TAT)-dependent signal peptide.

[0044] In some embodiments, the signal peptide is a Sec-dependent signal peptide selected from LamB, LTB, MalE, OmpA, PelB, PhoA, SpA, PhoE, OmpT, OmpF, and functional fragments or derivatives thereof.

[0045] In some embodiments, the signal peptide is a PelB signal peptide or a functional fragment or functional fragment or derivative thereof. [0046] In some embodiments, the PelB signal peptide, or the functional fragment or functional fragment or derivative thereof, comprises the amino acid sequence MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 1).

[0047] In some embodiments, the signal peptide is a TAT-dependent signal peptide selected from TorA, Tap, and functional fragments or derivatives thereof.

[0048] In some embodiments, the signal peptide is a TorA signal peptide or a functional fragment or functional fragment or derivative thereof.

[0049] In some embodiments, the TorA signal peptide, or the functional fragment or functional fragment or derivative thereof, comprises the amino acid sequence MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 2).

[0050] In some embodiments, expression of the fusion polypeptide is under control of a promotor.

[0051] In some embodiments, the promoter is a constitutive promoter.

[0052] In some embodiments, the promoter is an inducible promoter.

[0053] In some embodiments, the inducible promoter is a modified T7 promoter.

[0054] In some embodiments, the modified T7 promoter comprises a sequence selected from TAATACGACTCACTAATACTGAA (SEQ ID NO: 3),

TAATACGACTCACTATTTCGGAA (SEQ ID NO: 4),

TAATACGACTCTCTATAGG (SEQ ID NO: 5), TAATACGACTCACTATAGGAGAA (SEQ ID NO: 6), TAATACCACTCACTATAGGGAGA (SEQ ID NO: 7), TAATACAACTCACTATAGGGAGA (SEQ ID NO: 8), and

TAATACGTCTCACTATAGGGGAA (SEQ ID NO: 9).

[0055] In some embodiments, the modified T7 promoter comprises the sequence TAATACGACTCTCTATAGG (SEQ ID NO: 5).

[0056] In some embodiments, the inducible promoter is an isopropyl [3-d-l- thiogalactopyranoside (IPTG)-inducible promoter.

[0057] In some embodiments, the IPTG-inducible promoter is induced in the presence of 0.01 mM-3.0 mM IPTG.

[0058] In some embodiments, the inducible promoter is a tetracycline (Tc)-inducible promoter.

[0059] In some embodiments, the tetracycline (Tc)-inducible promoter comprises the sequence GTTGACACTCTATCATTGATAGAGTTATTTTACCACTC (SEQ ID NO: 69).

[0060] In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of 1-1500 ng/ml anhydrotetracycline. [0061] In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of about 1000 ng/ml anhydrotetracycline.

[0062] In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of 1-1500 ng/ml tetracycline.

[0063] In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of about 1000 ng/ml tetracycline.

[0064] In some embodiments, the first polynucleotide and/or the additional polynucleotide(s) is integrated into a cellular chromosome.

[0065] In some embodiments, the first polynucleotide is integrated into a cellular chromosome.

[0066] In some embodiments, the first polynucleotide and/or the additional polynucleotide(s) is present on a plasmid.

[0067] In some embodiments, the additional polynucleotide(s) is present on a plasmid.

[0068] In some embodiments, the fusion polypeptide comprises the same toxin component as the toxin component of the endogenous TA module of said cell, and the activity of the endogenous TA module of said cell is eliminated by deletion of the gene encoding the endogenous TA module.

[0069] In some embodiments, the TA module is MazEF and the toxin component is MazF toxin, or a functional fragment or functional fragment or derivative thereof.

[0070] In some embodiments, the MazF toxin, or the functional fragment or functional fragment or derivative thereof, comprises the amino acid sequence

MVSRYVPDMGDLIWVDFDPTKGSEQAGHRPAVVLSPFMYNNKTGMCLCVPCTTQS KGYPFEVVLSGQERDGVALADQVKSIAWRARGATKKGTVAPEELQLIKAKINVLIG (SEQ ID NO: 62).

[0071] In some embodiments, the first polynucleotide and/or the one or more additional polynucleotide(s) that are modified comprise the sequence Adenine-Cytosine- Adenine (ACA). [0072] In some embodiments, the ACA sequence(s) in the first polynucleotide and/or the one or more additional polynucleotide(s) is replaced such that the amino acid sequence(s) of the encoded polypeptide(s) is the same as when the ACA sequence(s) is not replaced.

[0073] In some embodiments, the biological substance is selected from biomass or its constituents, plasmid DNA, recombinant protein, peptide, amino acid, antibiotic, biosurfactant, biological fuel, organic acid, fatty acid, polyol, flavor, fragrance, polynucleotide, nucleotide, vitamin, pigment, sugar, polysaccharide, biopolymer, and plastic. [0074] In some embodiments, the expression of the fusion polypeptide in said cell results in an increased efficiency of substrate utilization by the cell as compared to a control cell which does not express the fusion polypeptide.

[0075] In some embodiments, the increased efficiency of substrate utilization is manifested in an increased production of biomass or a biological substance, or combination thereof, from the same amount of substrate utilized by the cell as compared to the control cell.

[0076] In some embodiments, the expression of the fusion polypeptide in said cell results in an increased production of biomass or a biological substance, or combination thereof, as compared to a control cell which does not express the fusion polypeptide.

[0077] In some embodiments, the expression of the fusion polypeptide in said cell results in a slower rate of substrate utilization by the cell as compared to a control cell which does not express the fusion polypeptide.

[0078] In some embodiments, the slower rate of substrate utilization is manifested in an increased duration of cellular growth.

[0079] In some embodiments, the fusion polypeptide possesses an enzymatic activity.

[0080] In another aspect is provided a genetically modified E. coli cell comprising a first polynucleotide encoding a fusion polypeptide comprising MazF toxin, or a functional fragment or functional fragment or derivative thereof, operably linked to a signal peptide pelB or TorA, or a functional fragment or functional fragment or derivative thereof, wherein expression of the fusion polypeptide is under the control of a modified T7 promoter or a tetracycline (Tc)- inducible promoter, and wherein the endogenous toxin-antitoxin (TA) module MazEF has been deleted.

[0081] In some embodiments, the MazF toxin, or the functional fragment or functional fragment or derivative thereof, comprises the amino acid sequence MVSRYVPDMGDLIWVDFDPTKGSEQAGHRPAVVLSPFMYNNKTGMCLCVPCTTQS KGYPFEVVLSGQERDGVALADQVKSIAWRARGATKKGTVAPEELQLIKAKINVLIG (SEQ ID NO: 62).

[0082] In some embodiments, the PelB signal peptide, or the functional fragment or functional fragment or derivative thereof, comprises the amino acid sequence MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 1).

[0083] In some embodiments, the TorA signal peptide, or the functional fragment or functional fragment or derivative thereof, comprises the amino acid sequence MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 2). [0084] In some embodiments, the modified T7 promoter comprises a sequence selected from TAATACGACTCACTAATACTGAA (SEQ ID NO: 3),

TAATACGACTCACTATTTCGGAA (SEQ ID NO: 4),

TAATACGACTCTCTATAGG (SEQ ID NO: 5), TAATACGACTCACTATAGGAGAA (SEQ ID NO: 6), TAATACCACTCACTATAGGGAGA (SEQ ID NO: 7), TAATACAACTCACTATAGGGAGA (SEQ ID NO: 8), and

TAATACGTCTCACTATAGGGGAA (SEQ ID NO: 9).

[0085] In some embodiments, the modified T7 promoter comprises the sequence TAATACGACTCTCTATAGG (SEQ ID NO: 5).

[0086] In some embodiments, the tetracycline (Tc)-inducible promoter comprises the sequence GTTGACACTCTATCATTGATAGAGTTATTTTACCACTC (SEQ ID NO: 69). [0087] In some embodiments, the first polynucleotide is integrated into a cellular chromosome.

[0088] In some embodiments, a genetically modified A. coli cell described herein may further comprise one or more additional polynucleotides, said additional polynucleotide(s) encoding at least one polypeptide which is a biological substance or which participates in the production of the biological substance, wherein the first polynucleotide encoding the fusion polypeptide and/or the additional polynucleotide(s) encoding the at least one polypeptide is modified to replace one or more ACA nucleotide sequences in the corresponding mRNA(s).

[0089] In some embodiments, the ACA sequence(s) in the first polynucleotide and/or the one or more additional polynucleotide(s) is replaced such that the amino acid sequence(s) of the encoded polypeptide(s) is the same as when the ACA sequence(s) is not replaced.

[0090] In some embodiments, the biological substance is selected from biomass or its constituents, plasmid DNA, recombinant protein, peptide, amino acid, antibiotic, biosurfactant, biological fuel, organic acid, fatty acid, polyol, flavor, fragrance, polynucleotide, nucleotide, vitamin, pigment, sugar, polysaccharide, biopolymer, and plastic.

[0091] In another aspect is provided a polynucleotide molecule comprising a polynucleotide sequence encoding a fusion polypeptide comprising a global metabolic regulator, or a component or functional fragment or functional fragment or derivative thereof, operably linked to a signal peptide which enables secretion of the fusion polypeptide to the space outside of the cytoplasmic membrane of a genetically modified cell upon expression of the fusion polypeptide is said cell.

[0092] In some embodiments, the expression of the fusion polypeptide in the genetically modified cell does not fully inhibit growth of said cell. [0093] In some embodiments, the growth rate of said genetically modified cell during the expression of the fusion polypeptide is higher than 0.

[0094] In some embodiments, the global metabolic regulator operably linked to the signal peptide is a toxin component of a toxin and antitoxin (TA) module, or a functional fragment or functional fragment or derivative thereof.

[0095] In some embodiments, the fusion polypeptide comprises the same toxin component, or the functional fragment or functional fragment or derivative thereof, as the toxin component of an endogenous TA module of said cell.

[0096] In some embodiments, a polynucleotide molecule described herein may further comprise one or more additional polynucleotide sequences, said additional polynucleotide sequence(s) encoding at least one polypeptide which is a biological substance or which participates in the production of the biological substance.

[0097] In some embodiments, the polynucleotide sequence encoding the fusion polypeptide and/or the one or more additional polynucleotide sequence(s) encoding the at least one polypeptide is modified to replace one or more nucleotide sequences in a corresponding mRNA(s) that are recognizable by said toxin component, or the functional fragment or functional fragment or derivative thereof, so that such mRNA(s) becomes resistant to the destruction by said toxin component.

[0098] In some embodiments, the genetically modified cell is a microbial cell.

[0099] In some embodiments, the microbial cell is a prokaryotic cell.

[00100] In some embodiments, the prokaryotic cell is a bacterial cell.

[00101] In some embodiments, the prokaryotic cell is from a genus selected from Nocardia, Acelobacler, Bacillus, Clostridium, Corynebacterium, Erwinia, Escherichia, Gluconacetobacter , Gluconobacter , Klebsiella, Lactococcus, Lactobacillus, Mannheimia, Propionibacterium, Pseudomonas, Ralstonia, Saccharopolyspora, Salmonella, Serratia, Streptomyces, Xanthomonas, and Zymomonas.

[00102] In some embodiments, the prokaryotic cell is from a species selected from Acetobacter aceti, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus subtilis, Clostridium acetobutylicum, Clostridium butyricum, Clostridium thermoaceticum, Clostridium tyrobutyricum, Corynebacterium glutamicum, Erwinia citreus, Erwinia herbicola, Gluconacetobacter hansenii, Gluconobacter oxydans, Klebsiella oxytoca, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Mannheimia succinicip-roducens, Nocardia lactamdurans, Propionibacterium shermanii, Pseudomonas denitrificans, Ralstonia eutropha, Saccharopolyspora erythrea, Saccharopolyspora spinosa, Serratia marcescens, Streptomyces clavuligerus, Streptomyces griseus, Streptomyces lividans, Streptomyces roseosporus, Xanthomonas campeslris. Zymomonas mobd ' is. Escherichia coH, Lactococcus lactis, Bacillus cere us. Salmonella typhi murium, and Pseudomonas fluor escens.

[00103] In some embodiments, the prokaryotic cell is from a species selected from Escherichia coH, Lactococcus lactis, Bacillus subliHs, Bacillus cercus, Salmonella typhi murium, and Pseudomonas fluor escens.

[00104] In some embodiments, the prokaryotic cell is from the species Escherichia coli.

[00105] In some embodiments, the signal peptide enables secretion of the fusion polypeptide to the periplasm of the microbial cell.

[00106] In some embodiments, the signal peptide enables secretion of the fusion polypeptide to the extracellular space of the microbial cell.

[00107] In some embodiments, the signal peptide is a Sec-dependent signal peptide, or a twin- arginine translocation (TAT)-dependent signal peptide.

[00108] In some embodiments, the signal peptide is a Sec-dependent signal peptide selected from LamB, LTB, MalE, OmpA, PelB, PhoA, SpA, PhoE, OmpT, OmpF, and functional fragments or derivatives thereof.

[00109] In some embodiments, the signal peptide is a PelB signal peptide, or a functional fragment or functional fragment or derivative thereof.

[00110] In some embodiments, the PelB signal peptide, or the functional fragment or functional fragment or derivative thereof, comprises the amino acid sequence MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 1).

[00111] In some embodiments, the signal peptide is a TAT-dependent signal peptide selected from TorA, Tap, and functional fragments or derivatives thereof.

[00112] In some embodiments, the signal peptide is a TorA signal peptide or a functional fragment or functional fragment or derivative thereof.

[00113] In some embodiments, the TorA signal peptide, or the functional fragment or functional fragment or derivative thereof, comprises the amino acid sequence MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 2).

[00114] In some embodiments, the polynucleotide sequence encoding the fusion polypeptide is operably linked to a promotor.

[00115] In some embodiments, the promoter is a constitutive promoter.

[00116] In some embodiments, the promoter is an inducible promoter.

[00117] In some embodiments, the inducible promoter is a modified T7 promoter. [00118] In some embodiments, the modified T7 promoter comprises a sequence selected from TAATACGACTCACTAATACTGAA (SEQ ID NO: 3),

TAATACGACTCACTATTTCGGAA (SEQ ID NO: 4),

TAATACGACTCTCTATAGG (SEQ ID NO: 5), TAATACGACTCACTATAGGAGAA (SEQ ID NO: 6), TAATACCACTCACTATAGGGAGA (SEQ ID NO: 7), TAATACAACTCACTATAGGGAGA (SEQ ID NO: 8), and

TAATACGTCTCACTATAGGGGAA (SEQ ID NO: 9).

[00119] In some embodiments, the modified T7 promoter comprises the sequence TAATACGACTCTCTATAGG (SEQ ID NO: 5).

[00120] In some embodiments, the inducible promoter is an isopropyl [3-d-l- thiogalactopyranoside (IPTG)-inducible promoter.

[00121] In some embodiments, the inducible promoter is a tetracycline (Tc)-inducible promoter.

[00122] In some embodiments, the tetracycline (Tc)-inducible promoter comprises the sequence GTTGACACTCTATCATTGATAGAGTTATTTTACCACTC (SEQ ID NO: 69) [00123] In some embodiments, the fusion polypeptide comprises the same toxin component, or the functional fragment or functional fragment or derivative thereof, as the toxin component of the endogenous TA module of said cell.

[00124] In some embodiments, the TA module is MazEF and the toxin component is MazF toxin, or a functional fragment or functional fragment or derivative thereof.

[00125] In some embodiments, the MazF toxin, or the functional fragment or functional fragment or derivative thereof, comprises the amino acid sequence MVSRYVPDMGDLIWVDFDPTKGSEQAGHRPAVVLSPFMYNNKTGMCLCVPCTTQS KGYPFEVVLSGQERDGVALADQVKSIAWRARGATKKGTVAPEELQLIKAKINVLIG (SEQ ID NO: 62).

[00126] In some embodiments, the one or more nucleotide sequences in the corresponding mRNA(s) that are recognizable by the toxin component of the TA module, or the functional fragment or functional fragment or derivative thereof, comprise the sequence Adenine- Cytosine- Adenine (AC A).

[00127] In some embodiments, the ACA sequence(s) in the corresponding mRNA(s) is replaced such that the amino acid sequence(s) of the encoded polypeptide(s) is the same as when the ACA sequence(s) is not replaced.

[00128] In some embodiments, the biological substance is selected from biomass or its constituents, plasmid DNA, recombinant protein, peptide, amino acid, antibiotic, biosurfactant, biological fuel, organic acid, fatty acid, polyol, flavor, fragrance, polynucleotide, nucleotide, vitamin, pigment, sugar, polysaccharide, biopolymer, and plastic.

[00129] In another aspect provided is a polynucleotide molecule comprising a polynucleotide sequence encoding a fusion polypeptide comprising MazF toxin, or a functional fragment or functional fragment or derivative thereof, operably linked to a signal peptide pelB or TorA, or a functional fragment or functional fragment or derivative thereof, wherein the polynucleotide sequence encoding the fusion polypeptide is optionally operably linked to a modified T7 promoter or a tetracycline (Tc)-inducible promoter.

[00130] In some embodiments, the MazF toxin, or the functional fragment or functional fragment or derivative thereof, comprises the amino acid sequence MVSRYVPDMGDLIWVDFDPTKGSEQAGHRPAVVLSPFMYNNKTGMCLCVPCTTQS KGYPFEVVLSGQERDGVALADQVKSIAWRARGATKKGTVAPEELQLIKAKINVLIG (SEQ ID NO: 62).

[00131] In some embodiments, the PelB signal peptide, or the functional fragment or functional fragment or derivative thereof, comprises the amino acid sequence MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 1).

[00132] In some embodiments, the TorA signal peptide, or the functional fragment or functional fragment or derivative thereof, comprises the amino acid sequence MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 2).

[00133] In some embodiments, the modified T7 promoter comprises a sequence selected from TAATACGACTCACTAATACTGAA (SEQ ID NO: 3),

TAATACGACTCACTATTTCGGAA (SEQ ID NO: 4),

TAATACGACTCTCTATAGG (SEQ ID NO: 5), TAATACGACTCACTATAGGAGAA (SEQ ID NO: 6), TAATACCACTCACTATAGGGAGA (SEQ ID NO: 7), TAATACAACTCACTATAGGGAGA (SEQ ID NO: 8), and

TAATACGTCTCACTATAGGGGAA (SEQ ID NO: 9).

[00134] In some embodiments, the modified T7 promoter comprises the sequence TAATACGACTCTCTATAGG (SEQ ID NO: 5).

[00135] In some embodiments, the tetracycline (Tc)-inducible promoter comprises the sequence GTTGACACTCTATCATTGATAGAGTTATTTTACCACTC (SEQ ID NO: 69). [00136] In some embodiments, a polynucleotide molecule described herein may further comprise one or more additional polynucleotide sequences, said additional polynucleotide sequence(s) encoding at least one polypeptide which is a biological substance or which participates in the production of the biological substance, wherein the polynucleotide sequence encoding the fusion polypeptide and/or the additional polynucleotide sequence(s) encoding the at least one polypeptide is modified to replace one or more ACA nucleotide sequences in a corresponding mRNA(s).

[00137] In some embodiments, the ACA sequence(s) in the corresponding mRNA(s) is replaced such that the amino acid sequence(s) of the encoded polypeptide(s) is the same as when the ACA sequence(s) is not replaced.

[00138] In some embodiments, the biological substance is selected from biomass or its constituents, plasmid DNA, recombinant protein, peptide, amino acid, antibiotic, biosurfactant, biological fuel, organic acid, fatty acid, polyol, flavor, fragrance, polynucleotide, nucleotide, vitamin, pigment, sugar, polysaccharide, biopolymer, and plastic.

[00139] In some embodiments, the fusion polypeptide possesses an enzymatic activity.

[00140] In another aspect is provided a recombinant construct comprising a polynucleotide molecule described herein.

[00141] In some embodiments, the construct is a plasmid.

[00142] In another aspect is provided a polynucleotide molecule comprising a modified T7 promoter comprising a sequence selected from TAATACGACTCACTAATACTGAA (SEQ ID NO: 3), TAATACGACTCACTATTTCGGAA (SEQ ID NO: 4), TAATACGACTCTCTATAGG (SEQ ID NO: 5), TAATACGACTCACTATAGGAGAA (SEQ ID NO: 6), TAATACCACTCACTATAGGGAGA (SEQ ID NO: 7), TAATACAACTCACTATAGGGAGA (SEQ ID NO: 8), and

TAATACGTCTCACTATAGGGGAA (SEQ ID NO: 9).

[00143] In some embodiments, the modified T7 promoter comprises the sequence TAATACGACTCTCTATAGG (SEQ ID NO: 5).

[00144] In another aspect is provided a recombinant construct comprising a polynucleotide molecule described herein.

[00145] In another aspect is provided a method of producing a biological substance, comprising culturing a genetically modified cell described herein under conditions suitable for producing the biological substance, and optionally purifying the biological substance.

[00146] In another aspect is provided a method of increasing efficacy of substrate utilization by a cell, said method comprising genetically modifying said cell by introducing a polynucleotide molecule described herein or a recombinant construct described herein.

[00147] In another aspect is provided a method of increasing production of biomass or a biological substance, or a combination thereof, by a cell, said method comprising genetically modifying said cell by introducing a polynucleotide molecule described herein or a recombinant construct described herein into the cell.

[00148] These and other aspects and embodiments described herein will be apparent to those of ordinary skill in the art in the following description, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[00149] Figure 1 shows a schematic representation of T448pelBmazF-KanR construction (1765bp) for homologous recombination.

[00150] Figure 2 shows a schematic representation of T448torAmazF-KanR construction (1827bp) for homologous recombination.

[00151] Figure 3 shows a schematic representation of tc_pelBmazF construction (1170bp) for homologous recombination.

[00152] Figure 4 shows a map of expression plasmid pet32a-hPTH.

[00153] Figure 5A shows the time course of E. coli T7 Express A growth in M9 mineral medium supplemented with 10 g/L glucose. The vertical arrow marks the moment of PelBMazF synthesis induction by addition of [3-d-l -thiogalactopyranoside (IPTG) to a final concentration of: 0.01 mM (■); 0.1 mM (A); 0.5 mM (x); 1.0 mM (•). E. coli T7 Express Iq (♦) was used as a control.

[00154] Figure 5B shows the time course of E. coli T7 Express Iq (♦), E. coli AT7 Exp del T448mazF-kanR (A) and E. coli T7 Express A (■) growth in M9 mineral medium supplemented with 10 g/L glucose. The vertical arrow marks the moment of PelBMazF synthesis induction by addition of [3-d- 1 -thiogalactopyranoside (IPTG) to a final concentration of 0.01 mM to the cultures of E. coli AT7 Exp del T448mazF-kanR and E. coli T7 Express A. All curves represent the mean average of three independent experiments.

[00155] Figure 6A shows the time course of E. coli T7 Express B growth in M9 mineral medium supplemented with 10 g/L glucose. The vertical arrow marks the moment of TorAMazF synthesis induction by addition of [3-d- 1 -thiogalactopyranoside (IPTG) to a final concentration of: 0 mM (■); 0.01 mM (A); 0.1 mM (♦); 0.5 mM (x); 1.0 mM (•). E. coli T7 Express Iq (+) was used as a control.

[00156] Figure 6B shows the time course of E. coli T7 Express Iq (dashed line) and E. coli T7 Express B (solid line) growth in M9 mineral medium supplemented with 10 g/L glucose. The vertical arrow marks the moment of TorAMazF synthesis induction by addition of [3-d- 1 - thiogalactopyranoside (IPTG) to a final concentration of 0.01 mM to the cultures of E. coli T7 Express B. All curves represent the mean average of three independent experiments. [00157] Figure 7 shows the time course of E. coli T7 Express C growth in M9 medium supplemented with 10 g/L glucose. The horizontal arrow marks the moment of PelBMazF synthesis induction by addition of ATc (solid lines) to a final concentration of: 500 ng/ml (♦); 800 ng/ml (■); 1000 ng/ml (•); 1500 ng/ml (A); 2000 ng/ml (▼). E. coli T7 Express Iq (dashed line) was used as a control. All curves represent the mean average of three independent experiments.

[00158] Figures 8A-8B shows the time course of E. coli T7 Express C growth in LB medium. The horizontal arrows mark the moment of PelBMazF synthesis induction by addition of ATc (solid line) to a final concentration of: 500 ng/ml (Figure 8A); 1000 ng/ml (Figure 8B). E. coli T7 Express Iq (dashed line) was used as a control. All curves represent the mean average of three independent experiments.

[00159] Figure 9 shows the time course of E. coli T7 Express A pet32a-hPTH (•) and E. coli T7 Express Iq pet32a-hPTH (A) growth in LB medium and hPTH expression ratio between the two strains (hPTH relative expression, bars). The vertical arrow marks the moment of hPTH (and PelBMazF in E. coli T7 Express A pet32a-hPTH) synthesis induction by addition of P-d- 1 -thiogalactopyranoside (IPTG) to a final concentration of 1.0 mM. The samples were taken after (a) 5.5 h growth (4 h induction) and (b) 7.5 h growth (6 h induction).

[00160] Figure 10A shows the time course of E. coli MG1655 growth in Luria-Bertani (LB) medium: ODeoo (♦) and specific growth rate p (bars).

[00161] Figure 10B shows the relative expression levels (right axis) of mcizF (♦) and mazE (A) and specific growth rate p (bars; left axis). The growth rate p was calculated as p = (ln[X]~ ln[Xo])/T, where T is time, and Xo and X are optical densities of the bacterial culture at time points zero and T, respectively. All points represent the mean average of triplicate spots; error bars denote standard deviation.

DETAILED DESCRIPTION

[00162] The present invention provides, among other things, compositions and methods for production of various biological substances. In particular, compositions and methods of the present invention relate to a genetically modified cell that may be useful for production any of various biological substances disclosed herein. The genetically modified cell may comprise a polynucleotide encoding a fusion polypeptide comprising a global metabolic regulator or a component or functional fragment or derivative thereof, e.g., a toxin component of a toxin and antitoxin (TA) module (e.g., a microbial TA module), which may be operably linked to a signal peptide. The signal peptide may enable the secretion of the fusion polypeptide to a space outside of the cytoplasmic membrane the cell, e.g., the periplasm and/or the extracellular space. In certain aspects, the genetically modified cell may comprise one of more additional polynucleotides encoding at least one polypeptide, which may be a biological substance or may participate in the production of a biological substance of the present disclosure. In various embodiments, e.g., when the global metabolic regulator operably linked to the signal peptide is a toxin component of a TA module, the polynucleotide encoding the fusion polypeptide and/or the additional polynucleotide(s) encoding the at least one polypeptide may be modified to replace and/or remove one or more nucleotide sequence(s) in the corresponding mRNA(s) that may be recognizable by the toxin.

[00163] In some embodiments, methods of the disclosure may include genetic modification of a cell, e.g., a microbial cell, by introducing to the cell polynucleotide(s) encoding a fusion polypeptide comprising a global metabolic regulator or a component or functional fragment or derivative thereof (e.g., a toxin component of a toxin-antitoxin (TA) module) and/or biological substances or related proteins; expressing these proteins from promoters, e.g., an constitutive promoter or an inducible promotor; and methods for optimized production of a biological substance.

[00164] In certain aspects, the present invention provides a method of production of useful biological substances by creating a flux of global metabolic regulator(s) of proteinaceous nature from the cytoplasm to the periplasm and/or to the extracellular space in order to stabilize intracellular concentration(s) of the global metabolic regulator(s) at levels optimal for production of a particular target biological substance. More specifically, the invention involves construction, cloning, and heterological expression of nucleic acid sequences encoding a fusion polypeptide comprising a signal peptide and a peptide of global metabolic regulator, which, being produced, may be secreted across the cytoplasmic membrane. In some embodiments, the fusion polypeptide may possess an enzymatic activity. In some embodiments, when a fusion polypeptide described herein possesses an enzymatic activity, the enzymatic activity may be inherent to the fusion polypeptide. In some embodiments, the fusion polypeptide does not possess an enzymatic activity.

[00165] In some embodiments, the present invention provides several applications of secretable global metabolic regulators, or components or derivatives thereof, (e.g., MazF) for development of complex phenotypes as metabolic backgrounds for further engineering of industrial microorganisms for large-scale production of useful biological substances by means of techniques and tools of systems metabolic engineering (Lee and Kim, 2015; Song et al., 2015; U.S. Patent Application Publication No. 2009/0124012; Suzuki et al. 2005).

[00166] In some embodiments, complex phenotype development with the use of a secretable global metabolic regulator presented herein is heterological expression in microbial cells of a nucleic acid sequence encoding a fusion polypeptide comprising (or consisting of) a signal peptide and the toxin MazF (a global metabolic regulator). Development of the secretable MazF according to the present invention includes, for example, expression of the polynucleotide encoding MazF fused to various signal sequences from constitutive or inducible promoters on plasmid vectors or chromosome, engineering recombinant proteins, and fermentations.

[00167] Microbial metabolism is a conservative process that typically does not expend energy or nutrients to make compounds already available in the environment and does not overproduce components of intermediary metabolism. Coordination of metabolic functions ensures that, at any given moment, only the necessary enzymes, and the correct amount of each, are made. Once a sufficient quantity of a material is made, the enzymes involved in its formation are no longer synthesized, and the activities of preformed enzymes are curbed by a number of specific regulatory mechanisms (Engstrom and Pfleger 2017; Sanchez and Demain, 2008). Conversely, a remarkable metabolic burden is imposed by overproduction of target substances through engineered pathways in cells with dysregulated metabolism (Wu et al., 2016). In addition to the aspects listed above, accumulation of high concentrations of the target substances in the culture broth is also a significant factor decreasing viability and production potential of cells (Departs et al., 2017).

[00168] Therefore overproducing industrial microorganisms are required to merge the growth, production, and tolerance phenotypes into one combining features of the three under conditions of large-scale fermentation processes. Such complex phenotypes of overproducing cells could be successfully established mainly by engineering at the top tier of the regulatory system of cellular metabolism since global patterns of gene expression determining each of these phenotypes are governed by corresponding global metabolic regulators (Lin et al., 2013; Mukhopadhyay, 2015; Si, 2014; Wehr et al., 2019).

[00169] The present inventors hypothesized that, since this results in disruption of naturally existing links between the sensory and the regulatory systems of the cell, simultaneous expression of the genes encoding these global metabolic regulators should be placed under control of separate promoters allowing synthesis of these proteins at the predetermined levels. The present inventors further hypothesized that intracellular concentrations of these proteins and their ratios, necessary to manifest the desired complex phenotype, can be maintained by establishing permanent outfluxes of these proteins in order to avoid their accumulation in the cytoplasm, e.g., through various secretory systems of the microorganisms.

[00170] To date, applications of secretion of recombinant proteins in the biotech/biopharma industry have been limited exclusively to the export of end products. It provides several advantages over intracellular production. These advantages include simplified downstream processing, enhanced biological activity, higher product stability and solubility, andN-terminal authenticity of the expressed peptide (Freudl, 2018). Due to this, the secretory production of a target protein can drastically decrease the overall costs of production (Quax, 1997). In Gramnegative bacteria such as E. coll. two pathways attributed to Type II secretion system (Secdependent and TAT) are most often used for secretion of heterologous proteins (Freudl, 2018; Kleiner-Grote et al., 2018; Mergulhao et al., 2005), whereas only the Sec-pathway is currently exploited for the secretion of heterologous proteins from Gram-positive Bacillus subtilis and its relatives (Pohl and Harwood, 2010). In eukaryotic microorganisms, proteins are translocated from the cytosol to the endoplasmic reticulum via two translocon pore complexes Sec61 and Sshl in combination with different channel partners (Conesa et al., 2001; Delic et al., 2013; Wang et al., 2020). Targeting of a recombinant protein to a particular secretion system is determined by the choice of a signal peptide for fusion with a target protein (Freudl, 2018; Kleiner-Grote et al., 2018; Mergulhao et al., 2005; Rosano and Ceccarelli, 2014). However, none of the described secreted recombinant proteins are metabolic regulators.

[00171] Prior to the discovery of the present invention, the predominant view of the natural biological function of the toxin-antitoxin (TA) module MazEF has been formation of the persister cells in response to environmental stresses. According to this concept, the unbound MazF induces a bacteriostatic condition in a subpopulation of persister cells in a bacterial population that exhibit tolerance to various environmental stress conditions because of phenotypic transition into a dormant state (Harms et al., 2018; Tripathi et al., 2014; Yamacuchi and Inouye, 2011). However, the results of the experiments disclosed in the Examples section herein (see, e.g., Example 5) suggest that the natural function of the MazEF TA module in microbial cells is as a metabolic switch to transition from the “feast” phenotype to the “hunger” one, where the term “feast” denotes the unlimited growth in the presence of nutrient excess (Koch, 1971) and the term “hunger” denotes the growth in the environment in which bacteria are limited for one or more classes of essential nutrient (Ferenci, 2001) (see also U.S. Patent Application US20090124012, the content of which is incorporated herein by reference in its entirety for all purposes). This growth is associated with the metabolic emphasis on scavenging many different residual nutrients with most catabolic pathways maximally expressed. Cells also activate biosynthetic functions to maintain growth at the highest possible rate (Baev et al., 2006; Harder and Dijkhuizen, 1983).

[00172] The conclusion presented in the instant application that the natural function of the MazEF TA module in E. coll cells is being a metabolic switch to transition from the “feast” phenotype to the “hunger” one is based on experimental observations disclosed herein of the exact coincidence of a profound peak in mazF expression with the 5-fold drop of the culture growth rate upon entry into the “hunger” state, which distinguishes mazF expression curve from all other E. coli MG1655 gene expression curves. The peak in mazF expression is flanked by two peaks in expression of the mazE gene (Figure 10). The comparison of the mazE and mazF expression curves suggests that synthesis of the corresponding two proteins are regulated in such a way that the inhibitory action of MazF is maximal when cells need to slow down their metabolism and rearrange it in accordance with the deteriorated nutritional status of the medium, and is attenuated by MazE when the rate of metabolic processes is adjusted to the environmental conditions. Thus, the naturally occurring expression of mazF does not usher cells into the dormant state, but rather, passing through the “hunger” state, into the stationary phase, which is not synonymous with dormancy. Indeed, the dormant state of a bacterial cell is defined as levels of metabolic activity that are undetectable under normal laboratory conditions (Dworkin J and Shah IM. Exit from dormancy in microbial organisms. Nat Rev Microbiol. 2010, 8(12): 890-896), whereas the stationary phase is the stage when growth ceases but cells remain metabolically active and able to produce new proteins (Gefen O, Fridman O, Ronin I, Balaban NQ. Direct observation of single stationary-phase bacteria reveals a surprisingly long period of constant protein production activity. Proc Natl Acad Sci U S A. 2014, 111 (1): 556-610).

[00173] The complex phenotype obtained as a result of expression of secretable MazF in microbial cells according to the present invention combines traits of both the “feast” phenotype and the “hunger” phenotype and is characterized by a significantly higher efficiency of substrate utilization and, in some embodiments, prolonged growth. This piece of genetic engineering lays a metabolic background for further manipulations with cellular metabolism in order to redirect metabolic fluxes from dissimilative to assimilative pathways with the aim to enhance production of biomass and/or target products and/or decrease accumulation of undesired by-products. Thus, it presents a powerful tool for development and optimization of microbial strains for production of various biological substances. [00174] Expression of mazF from a strong inducible promoter inhibits cellular growth as a result of degradation of most cellular mRNAs and inhibition of protein synthesis (Christensen et al., 2003; Zhang et al., 2003b), but it does not affect DNA and RNA synthesis, indicating that metabolic activities necessary for ATP production and nucleotide biosynthesis are retained in the cells overproducing MazF (Suzuki et al., 2005). Subsequent overexpression of mazE restores protein synthesis in the cell (Christensen et al., 2003). Recent studies suggest that, even when mazF is expressed from a weak promoter, unbound MazF molecules may gradually accumulate in the cell to the growth-inhibiting concentrations when the MazE protein is not synthesized proportionally (Nikolic et al., 2017; 2022), bringing to light a possible challenge underlying the use of stable toxins in metabolic engineering.

[00175] Single Protein Production (SPP) system is based on the idea that E. coli cells transformed to overproduce MazF are able to produce mostly proteins, which genes have been engineered to alter all the AC A sequences in the corresponding mRNAs to non-MazF- cleavable sequences. This modification does not affect the amino acid sequence of the synthesized proteins due to the general codon degeneracy (Suzuki et al., 2005; US Patent Nos. 7,985,575; 9,499,825, each of which is incorporated herein by reference in its entirety). The recombinant proteins are produced at a level of up to 30% of the total cellular protein with no background cellular protein synthesis. The cells are metabolically active and synthesize the recombinant proteins for more than seven days. Nevertheless, the SPP system has limited industrial application as it can be used for manufacturing only very small amounts of prohibitively expensive proteins labeled with isotopes such as ¹⁵N and ¹³C or toxic amino acid analogs without labeling any other cellular proteins (Suzuki et al., 2005; US Patents Nos. 9,228,217; 9,328,368; 10,131,915, each of which is incorporated herein by reference in its entirety). The MazF overexpression from a strong promoter used in the SPP system results in a complete shutdown of native-protein synthesis and arrest of cell growth, whereas the conventional bio-manufacturing is mainly based on the use of growing cells. Other applications of MazF are described in, for example, US Patent Nos. 9,637,736, 8,975,061, 10,718,001, 10,696,997, 9,309,518, and 8,470,580, each of which is incorporated herein by reference in its entirety.

[00176] Engineering the mazF gene for production of a secretable MazF protein and its expression in cells including microbial cells as described herein result in establishing intracellular concentration of the unbound toxin at the level sufficient to regulate the rate of protein synthesis, the metabolic activity of the cell, the rate of substrate utilization, and cellular growth but not sufficient to arrest them. The ability of MazF and toxin components of other TA modules engineered for secretion, to regulate but not arrest protein synthesis and cellular metabolism constitute powerful tools for metabolic engineering for strain development and industrial fermentations.

[00177] Maintaining intracellular concentrations of unbound MazF at the level sufficient to modulate the rate of protein synthesis, metabolic activity of the cell, the rate of substrate utilization, and cellular growth but not sufficient to arrest them, creates a possibility to achieve a balance between dissimilative and assimilative branches of cellular metabolism. The ensued improvements, which eliminate some drawbacks of current microbial fermentation processes are increased yield of biomass by utilized substrate, decreased accumulation of toxic byproducts, decreased oxygen consumption, decreased heat generation, performing cultivation of microorganisms at higher nutrient concentrations. This application of secreted toxin components of TA modules embodies a technique for development of high efficiency and costeffectiveness microbial fermentation processes.

[00178] Expression of homo- or heterologous genes, modified to transcribe into non- cleavable-by-mRNA-interferase mRNAs according to the method employed in the SPP system (Suzuki et al., 2005; US Patent Nos. 7,985,575 and 9,499,825, each of which is incorporated herein by reference in its entirety), in cells producing secretable MazF, allows redirecting cellular resources to the synthesis of proteins encoded by the modified genes without arrest of cell growth. This application of secreted toxin components of TA modules embodies a technique for development of new microbial strains overproducing recombinant proteins in a growth-associated manner.

[00179] The same alteration of genes coding for key rate-limiting enzymes of particular metabolic pathways makes these pathways insensitive to the inhibition by mRNA interferase. On the contrary, alteration of “metabolic” genes to transcribe into a cleavage-sequence- enriched mRNAs (AC A, in the case of MazF) makes synthesis of the corresponding enzymes more susceptible to the inhibition by mRNA interferase and suppresses functioning of the metabolic pathway consisting of these enzymes. This application of secreted toxin components of TA modules embodies a technique for development of a metabolic background favorable for redirecting cellular resources to production of target metabolites in a growth-associated manner.

[00180] Fermentation processes designed according to the present invention are distinguished from the currently implemented ones by increased levels of both the target substance production and the product (target substance) yield by utilized substrate. These applications of secreted toxin components of TA modules pertain to virtually any living cell (both prokaryotic and eukaryotic) because of the universality of the genetic code (Shimazu et al., 2007; 2014).

Definitions

[00181] As used herein, the indefinite articles "a", "an" and "the" should be understood to include plural reference unless the context clearly indicates otherwise.

[00182] The term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.

[00183] The term “biological substance” as used herein refers to any of various substances comprising any of presently or previously living organism(s), or parts of such organisms or products thereof in their natural or modified forms. In certain embodiments, a biological substance may encompass biomass or its constituents, and/or products of biosynthesis localized either intracellularly or extracellularly. As a non-limiting example, the biological substance may be, e.g., a recombinant protein, peptide, amino acid, enzyme, antibiotic, biosurfactant, biological fuel, organic acid, fatty acid, polyol, flavor, fragrance, polynucleotide, nucleotide, vitamin, pigment, sugar, polysaccharide, biopolymer, or plastic. As another non-limiting example, the biological substance may be plasmid DNA.

[00184] The term “global metabolic regulator” refers to any molecule that may define certain pleiotropic phenotypes by coordinately controlling one or more operons. The operon(s) may be distributed throughout the genome, and/or may represent any number of disparate functions. In some embodiments, a global metabolic regulator may influence cellular morphology and/or cellular metabolic fluxes at the transcriptional and/or post-transcriptional levels.

[00185] “Operably linked” as used herein refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. In the context of the present invention, it refers, for example, to the relationship between a nucleic acid segment encoding a signal peptide and a nucleic acid segment encoding a toxin component of a TA module, wherein the signal peptide and the toxin component form a fusion protein. The term “operably linked” also refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, z.e., they are cisacting. However, some transcriptional regulatory sequences such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

[00186] The terms “vector” and “construct” are used interchangeably and comprises a nucleic acid that can infect, transfect, or transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein and/or lipid. The term encompasses both expression and non-expression vectors. Large numbers of suitable vectors are known to those of skill in the art and are commercially available. Where a recombinant microorganism is described as hosting a vector this includes both extra-chromosomal circular and linear DNA and DNA that has been incorporated into the host genome. Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome. Low copy number or high copy number vectors may be employed with the present invention.

[00187] As used herein, the term “expression vector” refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which may facilitate the expression of a polypeptide coding sequences in a host cell. Expression vectors may comprise, without limitation, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for particular hosts, including prokaryotic and eukaryotic hosts of the disclosure. Expression vectors may comprise one or more selectable marker genes to permit selection of host cells containing the vector. Such selectable markers include, without limitation, genes conferring, e.g., antibiotic resistance in bacteria (e.g., tetracycline or ampicillin resistance in A. co l , and the S. cerevisiae TRP1 gene.

[00188] As used herein, the term “promoter” includes all sequences capable of driving transcription of a coding sequence in a cell. Thus, promoters used in the vectors of the invention include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) transcription. “Constitutive” promoters are those that drive expression continuously under most environmental conditions and states of development or cell differentiation. “Inducible” or “regulatable” promoter direct expression of the nucleic acid of the invention under the influence of environmental conditions or developmental conditions. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light.

[00189] The terms “polypeptide,” “peptide” or “protein” are used interchangeably to refer to polymeric forms of amino acids of any length, including chemically or biochemically modified or derivatized amino acids. The terms encompass all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP- ribosylation, pegylation, biotinylation, etc.).

[00190] The terms “nucleic acid” and “polynucleotide,” used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. They include single-, double-, and multi -stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized polynucleotides.

[00191] As used herein, the term “derivative” in the context of proteins or polypeptides encompasses all mutated and/or modified versions of the proteins or polypeptides, and/or components thereof. As a non-limiting example, a polypeptide may be modified by altering its amino acid sequence via changing the polypeptide itself or the nucleic acid encoding the polypeptide. As a non-limiting example, a polypeptide may be modified such that the polypeptide has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the polypeptide of which it is a derivative. Derivatives of the amino acid sequence may include, without limitation, insertions, additions, deletions, and/or substitutions of one or more of any of various amino acids comprising the polypeptide. As a non-limiting example, a polypeptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations relative to the polypeptide of which it is a derivative.

[00192] As used herein, the term "isolated” means that the material (e.g., a nucleic acid, a polypeptide, a cell) is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.

[00193] As used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative definition.

[00194] As used herein, the term "recombinant" includes reference to a cell or vector that has been modified by the introduction of a heterologous nucleic acid sequence. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all as a result of a deliberate intervention.

[00195] The term "signal sequence" or "signal peptide" refers to any sequence of amino acids that participates in the effectuation of the secretion of a protein. This definition of the signal sequence is a functional one, meant to include all those amino acid sequences encoded by any portion of a protein gene. Signal peptides are often, but not universally, bound to the N- terminal portion of a protein or to the N-terminal portion of a precursor protein. The signal sequence may be endogenous/homologous (i.e., that normally associated with the protein (e.g., protease)), or may be exogenous/heterologous (i.e., from another secreted protein). As used herein, the term “signal sequence” also refers to nucleotide sequences encoding a signal peptide.

[00196] As used herein, the term “microbial cell” as used herein refers to any of various cells of organisms belonging to the kingdom Protista, which includes eukaryotes such as algae, fungi and protozoa, and prokaryotes such as eubacteria and archaebacteria (see, e.g., H.G. Schlegel, General Microbiology, Seventh Edition (1992) Cambridge University Press, p. 2.). [00197] In accordance with the present invention there may be employed conventional pharmacology and molecular biology techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein “Sambrook el al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (MJ. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985)); Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984)); Animal Cell Culture (R.I. Freshney, ed. (1986); Immobilized Cells and Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel el al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994); among others.

Genetically Modified Cells

[00198] In one aspect, the invention provides a genetically modified cell comprising a first polynucleotide encoding a fusion polypeptide comprising a global metabolic regulator, or a component or functional fragment or derivative thereof, operably linked to a signal peptide which may enable secretion of the fusion polypeptide to the space outside of the cytoplasmic membrane of said cell.

[00199] In various embodiments, the expression of the fusion polypeptide in the cell may not inhibit the growth of the cell. In various embodiments, the expression of the fusion polypeptide in the cell may not fully inhibit the growth of the cell. In some embodiments, the growth rate (e.g., specific growth rate) of the cell during the expression of the fusion polypeptide may be higher than 0 h’¹. As a non-limiting example, the growth rate may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 h . In certain embodiments, the expression of the fusion polypeptide in the cell may not inhibit the growth of the cell such that the cell is actively dividing. In certain embodiments, the expression of the fusion polypeptide in the cell may not fully inhibit the growth of the cell such that the cell is actively dividing. In some embodiments, the expression of the fusion polypeptide in the cell may not inhibit the growth of the cell such that the cell is proliferating. In some embodiments, the expression of the fusion polypeptide in the cell may not fully inhibit the growth of the cell such that the cell is proliferating

[00200] Metabolic regulation such as that which may occur, for example, via a global metabolic regulator may comprise any numbers of transcriptional factors. In some embodiments, the transcription factors may participate indirectly or directly in the regulation of operons or sets of operons that may comprise various metabolic pathways. In certain embodiments, such transcription factors may be considered global regulators, e.g., global metabolic regulators. Non-limiting examples of global metabolic regulators which are transcriptional factors that may regulate the expression of genes in E. coli include CRP, FNR, IHF, FIS, ArcA, NarL, and Lrp. Additional non-limiting examples of transcription factors include NarL, Fur, Mlc, CspA, Rob, PurR, PhoB, CpxR, SoxR/SoxS, OxyR, PdhR, ModE, FlhA, CysB, DnaA, BolA, and IciA. [00201] In some embodiments, the global metabolic regulator may act at the transcriptional level. In some embodiments, the global metabolic regulator may act at the post-transcriptional level. A non-limiting example of a global metabolic regulator that may act at the transcriptional level is sigma factors. Sigma factors may reversibly bind the core subunit of RNA polymerase (RNAP) to endow promoter specificity on the polymerase holoenzyme, thus mediating transcription of all genes in a prokaryotic cell, such as, but not limited to, bacteria (e.g., E. colt). Sigma factors may include, without limitation, E. coll sigma factors such as, such as but not limited to, c70 (oD), o54 (oN), c38 (oS), o32 (cH), and c24 (cE).

[00202] In some embodiments, the global regulator, e.g., global metabolic regulator, may act at the post-transcriptional level. In some embodiments, such global regulators may comprise, without limitation, Hfq, CsrA (Nogueira and Springer, 2000), Obg (Starosta et al., 2014) and toxin and antitoxin (TA) modules.

[00203] In some embodiments, any of the various genetically modified cells disclosed herein may comprise a first polynucleotide encoding a fusion polypeptide comprising a global metabolic regulator, or a component or functional fragment or derivative thereof. In some embodiments, the global metabolic regulator may be operably linked to a signal peptide. In some embodiments, the global metabolic regulator may comprise a toxin and antitoxin (TA) module, or a component or functional fragment or derivative thereof. In some embodiments, the global metabolic regulator, or a component or functional fragment or derivative thereof, may comprise a toxin component of a TA module.

[00204] Without wishing to be bound by theory, TA modules are widespread in prokaryotic genomes (Gerdes et al. 2005; Harms et al., 2018; Horak and Tamman 2017; Van Melderen 2010; Yamaguchi and Inouye, 2011). The E. coli K-12 MG1655 strain has at least 37 TA loci. The toxins expressed from TA loci use a wide variety of molecular activities to interfere with such cellular functions as replication, translation, and cell wall synthesis to inhibit bacterial growth. They are capable of cleaving, degrading, and/or modifying their cellular targets enzymatically, and thus may obstruct bacterial physiology even at low protein concentrations. Antitoxins are proteins or RNAs that control their cognate toxins through direct interactions and through transcriptional and translational regulation of TA module expression. Accordingly, TA modules may be categorized into six different types, depending on how the antitoxin neutralizes expression and/or activity of the toxin (Harms et al. 2018). The major exemplary biological functions of TA modules are post-segregational killing (Gerdes et al., 1986), abortive infection (Dy et al., 2014), and persister formation/antibiotic tolerance (Harms et al., 2016). [00205] In some embodiments, the ratio of toxin: antitoxin intracellular concentrations may be used as a factor for regulating protein synthesis and cellular metabolism at the level of mRNA translation. The ability of the TA modules disclosed herein to regulate, but not arrest, protein synthesis and metabolism is a key factor for successful industrial fermentation, strain development and metabolic engineering.

[00206] In some embodiments, TA modules disclosed herein may be used to increase yield of biomass by utilized substrate, decrease accumulation of toxic by-products, decrease oxygen consumption, decrease heat generation, and/or perform cultivation of microorganisms at higher nutrient concentrations. In some embodiments, TA modules may be used to control all of the above-described factors, thereby allowing increases in the efficiency and cost-effectiveness of an industrial fermentation process.

[00207] In some embodiments, expression of endogenous or heterologous genes may be modified such that they are no longer targeted by interferase enzymes, e.g., gene expression may be modified such that the genes are transcribed into mRNAs that may not be cleaved by mRNA interferase, i.e., are “non-cleavable-by-mRNA-interferase”. This allows the cells to redirect cellular resources to the synthesis of these proteins without arrest of cell growth. In some embodiments, the same alteration of genes coding for key rate-limiting enzymes of particular metabolic pathways is made; this makes these pathways insensitive to the inhibition by an mRNA interferase. In some embodiments, metabolic genes are altered to transcribe into one or more cleavage-sequence-enriched mRNAs (e.g., ACA); this would make synthesis of the corresponding enzymes more susceptible to the inhibition by mRNA interferase and suppress functioning of the metabolic pathway consisting of these enzymes. In some embodiments, both mRNA interferase toxin and antitoxin genes may be expressed under inducible conditions and independently from each other.

[00208] In some embodiments, genetically modified cells disclosed herein may comprise a first polynucleotide encoding a fusion polypeptide comprising, for example, a toxin component of a TA module. Non-limiting examples of genes encoding a toxin component of a TA module include the yoeB, yafQ, mazF, relE, yeeV, and hip A genes. Since the YefM, DinJ, MazE, RelB, YeeU, and HipB proteins function as antitoxins for the YoeB, YafQ, MazF, RelE, YeeV, and HipA toxins, respectively, it is possible to delete both of the yefM and yoeB genes, the dinJ and yafQ genes, the mazE and mazF genes, the relB and relE genes, the yeeV and yeeU genes, and the hipA and hipB genes, respectively, in the genetically modified cells of the present disclosure. [00209] In some embodiments, the compositions and methods as provided herein may allow the manipulation of cellular metabolism at the level of translation in order to redirect metabolic fluxes to enhance production of a targeted cellular metabolite or to decrease accumulation of undesired by-products. In some embodiments, any of the genetically modified cells of the present invention may comprise a first polynucleotide comprising a component of a TA module, e.g., a mRNA interferase TA module. TA modules may be generated by methods of recombinant DNA technology, which are well known in the art and may include, e.g., expression MazF from different promoters (e.g., constitutive and/or inducible promoters) on plasmid vectors and cellular chromosome, engineering recombinant proteins so they lack mRNA interferase recognition sequences, and fermentation.

[00210] The compositions and methods as provided herein may attenuate expression of mRNA interf erases such as, for example, MazF, ChpBK, PemK, or Yach, by the use of a weak constitutive promoter, or may balance expression by a simultaneous over-expression of the cognate antitoxin (MazE, ChpBI, PemI, YdcD, respectively) from a separate constitutive or inducible promoter, which results in decreasing intracellular concentration of unbound mRNA interferase molecules to the levels which are able to decrease protein synthesis in the cell, but not sufficient to arrest it completely.

[00211] In some embodiments, any of the genetically modified cells of the present disclosure may comprise a fusion polypeptide disclosed herein. The fusion polypeptide may comprise the same toxin component as the toxin component of an endogenous TA module of the cell and the activity of the endogenous TA module of the cell may be eliminated and/or inactivated, for example, by deletion of said endogenous TA module. In various embodiments, the endogenous TA module of the cell may be eliminated and/or inactivated by deletion of the gene encoding the endogenous toxin component. In various embodiments, the activity of endogenous TA module of the cell may be eliminated and/or inactivated by deletion of the gene encoding the endogenous toxin component, and optionally also by deletion of the gene encoding the corresponding antitoxin component. In some embodiments, the activity of the endogenous TA module of the cell may be eliminated and/or inactivated by deletion of the gene encoding the endogenous toxin component and the gene encoding the endogenous antitoxin component. In some embodiments, the TA module may be MazF and MazE (i.e., MazEF), and the toxin component may be MazF, or a functional fragment or derivative thereof. In some embodiments, the TA module may be MazF and MazE (i.e., MazEF), and the antitoxin component may be MazE, or a functional fragment or derivative thereof. [00212] In one aspect, the invention provides a genetically modified cell, e.g., a microbial cell, for production of a biological substance, the cell comprising a first polynucleotide encoding a fusion polypeptide that may comprise a toxin component of a TA module, wherein the toxin component may be operably linked to a signal peptide which enables the secretion of the fusion polypeptide to a space outside of the cytoplasmic membrane of said cell, e.g., the periplasm and/or to the extracellular space of the cell such as a microbial cell. In some embodiments, the expression of the fusion polypeptide may be under the control of a constitutive or an inducible promoter. In some embodiments, the cell may comprise one or more additional polynucleotides encoding at least one polypeptide which is a biological substance or which participates in the production of said biological substance. In some embodiments, the first polynucleotide and/or the one or more additional polynucleotide(s) may be modified to remove one or more nucleotide sequences in the corresponding mRNA(s) that are recognizable by the toxin component of said TA module. In some embodiments, the first polynucleotide and/or the one or more additional polynucleotide(s) may be modified to replace one or more nucleotide sequences in a corresponding mRNA(s) that are recognizable by the toxin component, or the functional fragment or derivative thereof, so that such mRNA(s) becomes resistant to the destruction by said toxin component. In some embodiments, the genes encoding the endogenous TA module in the cells are deleted. In some embodiments, the endogenous TA module in the cells is eliminated.

[00213] In a related aspect, the invention provides a method of generating a genetically modified cell, e.g., a microbial cell, the method comprising introducing into the cell a first polynucleotide encoding a fusion polypeptide comprising a toxin component of a TA module, wherein the toxin component may be operably linked to a signal peptide which enables the secretion of the fusion polypeptide to a space outside of the cytoplasmic membrane, e.g., the periplasm and/or to the extracellular space of the cell (e.g., a microbial cell). In some embodiments, the polynucleotide sequence may further comprise a constitutive promoter sequence or an inducible promoter sequence for controlling expression of the fusion polypeptide. In some embodiments, one or more additional polynucleotide(s) encoding at least one polypeptide which is the biological substance or which participates in the production of the biological substance may be introduced into the cell. In some embodiments, the first polynucleotide and/or the one or more additional polynucleotide(s) may be modified to remove one or more nucleotide sequences in the corresponding mRNA(s) recognizable by the toxin component of said TA module. In some embodiments, the first polynucleotide and/or the one or more additional polynucleotide(s) may be modified to replace one or more nucleotide sequences in a corresponding mRNA(s) that are recognizable by the toxin component, or the functional fragment or derivative thereof, so that such mRNA(s) becomes resistant to the destruction by said toxin component. In some embodiments, the method may further comprise eliminating activity of the endogenous TA module in the cell.

[00214] In various embodiments, when a genetically modified cell of the present disclosure comprises a first polynucleotide encoding a fusion polypeptide comprising the same toxin component as the endogenous TA module of said cell, the endogenous TA module of the genetically modified cell may be eliminated. In some embodiments, the endogenous TA module in the cell may be eliminated by about 50% or more. The endogenous TA module in the cell may be eliminated by from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99% to about 100%.

[00215] In various embodiments, when a genetically modified cell of the present disclosure comprises a first polynucleotide encoding a fusion polypeptide comprising the same toxin component as the endogenous TA module of said cell, the activity of the toxin component of the endogenous TA module of the genetically modified cell may be eliminated. In some embodiments, the toxin component of the endogenous TA module in the cell may be eliminated by about 50% or more. The toxin component of endogenous TA module in the cell may be eliminated by from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99% to about 100%. In some embodiments, when the toxin component of the endogenous TA module in the cell may be eliminated, the antitoxin component of the endogenous TA module may additionally be eliminated in said cell. The antitoxin component of the endogenous TA module in the cell may be eliminated by from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99% to about 100%.

[00216] In some embodiments, the global metabolic regulator may comprise a TA module. By way of a non-limiting example, the TA module may comprise a microbial TA module, including, without limitation, a bacterial TA module, e.g., an E. coll TA module such as, but not limited to MazEF. In some embodiments, the TA module may comprise MazF and MazE, and the toxin component is MazF, or a functional fragment or derivative thereof. In some embodiments, the global metabolic regulator may be a TA module, and the TA module is MazE and MazF.

[00217] In some embodiments, the TA system useful for practicing the present invention may be an A. coli toxin-antitoxin (TA) system. The A. coli toxin-antitoxin (TA) module MazEF was identified as a post-transcriptional metabolic regulator which globally affects protein synthesis in response to a variety of different stress conditions (Sauert et al., 2016; Vesperet al., 2011; Zhang et al., 2003). Specifically, the A. coli toxin-antitoxin (TA) system comprises a labile antitoxin MazE and a stable toxin MazF, which is a sequence-specific endoribonuclease that preferentially cleaves mRNAs at the 3’ end of the first A base in an Adenine-Cytosine- Adenine (ACA) sequence in a ribosome-independent manner (Zhang et al., 2003b, 2005; US Patent Nos. 8,183,011; 9,243,234). These proteins are encoded by the genes mazE and mazF organized in an operon located downstream of the relA gene (Aizenman et al., 1996). During exponential growth one MazE dimer may form a stable TA complex with two MazF dimers and neutralize the toxin. This complex, along with the MazE protein is a repressor for the mazEF operon (Zhang et al., 2003a). Therefore, expression of the operon is strongly repressed (Marianovski et al. 2001) and the toxic effect of the mRNA interferase is not exerted under these conditions (Engelberg-Kulka et al., 2004; Gerdes et al., 2005). However, any environmental stress causing growth inhibition may lead to the degradation of the MazE antitoxin by ATP-dependent serine proteases ClpAP and Lon (Aizenman et al., 1996; Christensen et al., 2003) and release of the unbound MazF toxin in the cell. The released MazF may attack and cleave mRNAs, thus inhibiting protein synthesis and cellular growth (Inouye, 2006). These adverse environmental conditions may promote de-repression of the mazEF operon (Muthuramalingam et al. 2016).

[00218] In various embodiments, the TA module is MazF and MazE (MazEF) and the toxin component is MazF, or a functional fragment or derivative thereof. In some embodiments, MazF, or a functional fragment or derivative thereof, may comprise the amino acid sequence of

MVSRYVPDMGDLIWVDFDPTKGSEQAGHRPAVVLSPFMYNNKTGMCLCVPCTTQS KGYPFEVVLSGQERDGVALADQVKSIAWRARGATKKGTVAPEELQLIKAKINVLIG (SEQ ID NO: 62), or a variant thereof. In some embodiments, the nucleotide sequence encoding the MazF, or a functional fragment or derivative thereof, may comprise the nucleotide sequence set forth in SEQ ID NO: 63. In certain embodiments, the nucleotide sequence encoding the MazF, or a functional fragment or derivative thereof, may comprise the nucleotide sequence set forth in SEQ ID NO: 64.

[00219] In various embodiments, the TA module is MazF and MazE (MazEF) and the antitoxin component is MazE, or a functional fragment or derivative thereof. In some embodiments, MazE, or a functional fragment or derivative thereof, may comprise the amino acid sequence of

MH4SSVKRWGNSPAVRIPATLMQALNLNIDDEVKIDLVDGKLIIEPVRKEPVFTLAEL VNDITPENLHENIDWGEPKDKEVW (SEQ ID NO: 65), or a variant thereof. In some embodiments, the nucleotide sequence encoding the MazE, or a functional fragment or derivative thereof, may comprise the nucleotide sequence set forth in SEQ ID NO: 66.

[00220] In some embodiments, the TA module may comprise MazF and MazE and the toxin component may be MazF, or a functional fragment or derivative thereof. In some embodiments, the first polynucleotide may be integrated into the chromosome at any locus, such as but not limited to, the locus of an endogenous toxin and/or antitoxin genes.

[00221] In certain embodiments, when any of the global metabolic regulator(s) operably linked to a signal peptide may be a toxin component of a TA module such as, but not limited to, MazF, or a functional fragment or derivative thereof, the polynucleotide encoding the global metabolic regulator(s) operably linked to a signal peptide and/or one or more additional polynucleotide(s) encoding the at least one polypeptide may be modified to replace one or more nucleotide sequences in the corresponding mRNA(s) that are recognizable by said toxin component. In some embodiments, the polynucleotide encoding the global metabolic regulator(s) operably linked to a signal peptide and/or the additional polynucleotide(s) encoding the at least one polypeptide may be modified to replace the one or more nucleotide sequence(s) in the corresponding mRNA(s) that may be recognizable by the toxin with a sequence that is not recognizable (i.e., a non-recognizable sequence) by the toxin sequence. In some embodiments, the nucleotide sequences recognizable by the toxin component of the TA module may comprise the sequence Adenine-Cytosine-Adenine (ACA). In certain embodiments, the ACA sequences in the corresponding mRNA(s) may be replaced.

[00222] In some embodiments, the global metabolic regulator is a toxin component of a TA module, such as, but not limited to, MazE and MazF. In various embodiments, the TA module is MazF and MazE, and the toxin component is MazF, or a functional fragment or derivative thereof. In various embodiments, when the TA module is MazF and MazE, and the toxin component is MazF, or a functional fragment or derivative thereof, any of the polynucleotides encoding the global metabolic regulator(s) operably liked to a signal peptide and/or one or more additional polynucleotides encoding the at least one polypeptide may be modified to remove one or more nucleotide sequences in the corresponding mRNA(s) that are recognizable by the MazF, or a functional fragment or derivative thereof. In some embodiments, the nucleotide sequences recognizable by the MazF, or a functional fragment or derivative thereof, may comprise the sequence Adenine-Cytosine-Adenine (ACA). In certain embodiments, the ACA sequences in the corresponding mRNA(s) may be replaced.

[00223] In some embodiments, any of the nucleic acids comprising target genes disclosed herein have been engineered to alter all the ACA sequences in the corresponding mRNAs to non-MazF-cleavable sequences. Such modification does not affect the amino acid sequence of the synthesized proteins due to the general codon degeneracy. A skilled artisan can recognize that an ACA sequence in an mRNA is encoded by a TGT sequence in its corresponding gene. Therefore, one may identify TGT sequences in a target gene and replace the TGT sequences to achieve the desired alterations in its corresponding mRNA.

[00224] It is to be understood that nucleotides comprising the ACA sequences disclosed herein do not need to belong to the same codon, and can be distributed between, for example, two codons, such as is exemplified by the configuration XXA-CAX (SEQ ID NO: 72) or XAC- AXX (SEQ ID NO: 73), where X may be any nucleotide, e.g., adenine (A), cytosine (C), guanine (G), uracil (U), or thymine (T), and the ACA sequence participates in the coding of two amino acids. In some embodiments, the ACA sequence may be distributed between two codons. In some embodiments, the ACA sequence belongs to the same codon, i.e., a Threonine codon. As a non-limiting example, when the ACA sequence(s) is a Threonine codon, it may be replaced by an alternate Threonine codon, e.g., a Threonine codon selected from ACG, ACC, and ACT.

[00225] In some embodiments, the genetically modified cell of the present disclosure may be any of various eukaryotic cell such as, but not limited to, algae, fungi, and protozoa. In some embodiments, the eukaryotic cell may be a plant cell. In some embodiments, the eukaryotic cell may be an animal cell, e.g., a mammalian cell. In various embodiments, the genetically modified cell of the present disclosure may be a prokaryotic cell such as, but not limited to bacterial cells including eubacteria and archaea, e.g., archaebacterial cells.

[00226] In some embodiments, the genetically modified cell of the present disclosure may be a microbial cell. Without wishing to be bound by theory, the microbial cell may be any of microbial cells familiar to those skilled in the art such as, but not limited to, prokaryotic cells, such as bacterial cells, archaea cells, or eukaryotic cells such as fungal cells, including yeast cells. The selection of an appropriate microbial cell is within the abilities of those skilled in the art.

[00227] In some embodiments, the microbial cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is a bacterial cell from the Enterob acteriaceae family (as classified according to the taxonomy used in the NCBI (National Center for Biotechnology Information database), including, without limitation, bacteria of any species belonging to the genera Nocardia, Acetobacter, Bacillus, Clostridium, Corynebacterium, Enterobacter, Erwinia, Escherichia, Gluconoacetobacter, Gluconobacter, Klebsiella, Lactobacillus, Lactococcus, Mannheimia, Morganella, Pantoea, Photorhabdus, Propionibacterium, Providencia, Pseudomonas, Ralstonia, Saccharopolyspora, Salmonella, Serratia, Shigella, Streptomyces, Xanthomonas, Yersinia, and Zymomonas. In some embodiments, the bacterial cell is Escherichia coli (E. coli).

[00228] In some embodiments, the prokaryotic cell of the present disclosure may from a genus selected from Nocardia, Acetobacter, Bacillus, Clostridium, Corynebacterium, Erwinia, Escherichia, Gluconoacetobacter, Gluconobacter, Klebsiella, Lactobacillus, Lactococcus, Mannheimia, Propionibacterium, Pseudomonas, Ralstonia, Saccharopolyspora, Salmonella, Serratia, Streptomyces, Xanthomonas, and Zymomonas.

[00229] Other non-limiting examples of prokaryotic cells which can be used include, e.g., any species within the genera Bacillus, Streptomyces, Salmonella, Pseudomonas, and Staphylococcus, including, e.g., Lactococcus lactis, Bacillus subtilis, Bacillus cereus, Salmonella typhimurium, and Pseudomonas fluorescens.

[00230] In some embodiments, the prokaryotic cell is from a species selected from Acetobacter aceti, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus subtilis, Clostridium acetobutylicum, Clostridium butyricum, Clostridium thermoaceticum, Clostridium tyrobutyricum, Corynebacterium glutamicum, Erwinia citreus, Erwinia herbicola, Gluconacetobacter hansenii, Gluconobacter oxydans, Klebsiella oxytoca, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Mannheimia succinicip-roducens, Nocardia lactamdurans, Propionibacterium shermanii, Pseudomonas denitrificans, Ralstonia eutropha, Saccharopolyspora erylhrea. Saccharopolyspora spinosa. Serratia marcescens. Streptomyces clavuligerus, Streptomyces griseus. Streptomyces lividans, Streptomyces roseosporus. Xanthomonas campeslris. Zymomonas mobilis. Escherichia coH, Lactococcus taclis. Bacillus cere us. Salmonella typhi murium, and Pseudomonas fluor escens.

[00231] In some embodiments, the prokaryotic cell is from a species including, without limitation, Escherichia coli, Lactococcus lactis, Bacillus subtilis, Bacillus cereus, Salmonella typhimurium, and Pseudomonas fluor escens.

[00232] In some embodiments, the prokaryotic cell is from the species Escherichia coli.

[00233] In some embodiments, when the genetically modified cell of the present disclosure is a microbial cell, any of the signal peptides disclosed herein may enable the secretion of the fusion polypeptide to the periplasm of the microbial cell. In some embodiments, when the genetically modified cell of the present disclosure is a microbial cell, any of the signal peptides disclosed herein may enable the secretion of the fusion polypeptide to the extracellular space of the microbial cell.

[00234] In some embodiments, the genetically modified cell disclosed herein may be a eukaryotic cell such as, but not limited to, a fungal cell. In some embodiments, the fungal cell is from the genus Chrysosporium, Eremothecium (Ashbya), Rhizopus, Acremonium (Cephalosporium), Arxula, Aspergillus, Blakeslea, Candida, Fusarium, Ganoderma, Hansenula, Kluyveromyces, Mortierella, Mucor, Pachisolen, Penicillium, Phaffia, Pichia, Rhizopus, Saccharomyces, Schizosaccharomyces, Schwanniomyces, Tolypocladium, Trichoderma, Umbelopsis, Yarrowia, and Zygosaccharomyces . In some embodiments, the fungal cell is a yeast cell. As a non-limiting example, the yeast cell may be from a genus selected from Pichia, Saccharomyces, Schizosaccharomyces, and Schwanniomyces . In some embodiments, the yeast cell may be Pichia pastoris. In some embodiments, the yeast cell may be Saccharomyces cerevisiae. In some embodiments, the yeast cell may be Schizosaccharomyces pombe. In some embodiments, the yeast cell may be Pichia pastoris. In some embodiments, the fungal cell may be Aspergillis niger, Aspergillus oryzae, Aspergillus soyae, Aspergillus terreus, Penicillium notatum, Penicillium griseofulvin, Penicillium roqueforti, Penicillium candidum, Penicillium camemberti, Penicillium citrinum, Penicillium bilagi, Fusarium moniliforme, Tolypocladium inflatum, Rhizopus arhizus, Candida etchellsii, Candida versatilis, or Saccharomyces rouxii.

[00235] In various embodiments, the eukaryotic cell may be from a species selected from Acremonium chrysogenum, Arxula adeninivorans, Aspergillus awamori, Aspergillus chrysogenum, Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, Candida boidinii, Candida sorenensis. Blakeslea trispora, Chrysosporium lucknowense, Eremothecium (Ashbya) gossypii, Fusarium venenatum, Ganoderma hicidum. Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus. Mortierella alpine, Mucor miehei, Pachysolen lannophihis, Penicillium brevicompactum, Penicillium chrysogenum, Phaffia rhodozyma, Pichia melhanoUca, Pichia stipitis, Rhizopus oryzae, Trichoderma reesei. Umbelopsis isabeHina, Yarrowia lipolytica, Zygosaccharomyces baiHi, Pichia pasloris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe.

[00236] In some embodiments, the eukaryotic cell may be from a species selected from Pichia pastor is, Saccharomyces cerevisiae, and Schizosaccharomyces pombe.

[00237] Where appropriate, the genetically modified cells, e.g., microbial cells may be cultured in conventional nutrient media that may be modified as appropriate, for example, for activating promoters, e.g., when an inducible promoter is used to control expression of the fusion polypeptide disclosed herein, and/or selecting transformants and/or amplifying the genes of the invention. In some embodiments, the genetically modified cell may be cultured in a media as appropriate for optimized efficacy of a constitutive promoter of the present disclosure. In some embodiments, the genetically modified cell may be cultured in a media as appropriate for the optimized expression and/or production of any of various biological substances disclosed herein. As a non-limiting example, the biological substance may be biomass or its constituents, plasmid DNA, recombinant protein, peptide, amino acid, antibiotic, biosurfactant, biological fuel, organic acid, fatty acid, polyol, flavor, fragrance, polynucleotide, nucleotide, vitamin, pigment, sugar, polysaccharide, biopolymer, and plastic. In some embodiments, the biomass may be a microbial biomass. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter may be induced by appropriate means (e.g., temperature shift or chemical induction) and the cells may be cultured for an additional period to allow them to produce the desired polypeptide or fragment thereof.

[00238] In some embodiments, any of the genetically modified cells disclosed herein may optionally comprise one or more additional polynucleotide(s) encoding at least one polypeptide which is the biological substance or which participates in the production of the biological substance. As a non-limiting example, a biological substance may be biomass or its constituents, and/or products of biosynthesis localized either intracellularly or extracellularly. In some embodiments, the biological substance may be plasmid DNA. Additional examples of a biological substance, without limitation, are a recombinant protein, peptide, amino acid, enzyme, antibiotic, biosurfactant, biological fuel, organic acid, fatty acid, polyol, flavor, fragrance, polynucleotide, nucleotide, vitamin, pigment, sugar, polysaccharide, biopolymer and plastic. In some embodiments, the biological substance is localized intracellularly. In some embodiments, the biological substance is localized extracellularly, for example, in the extracellular space. In some embodiments, the biological substance is localized intracellularly. In some embodiments, the biological substance is localized in the cytoplasm. In some embodiments, the biological substance is localized in the periplasmic space. In some embodiments, the one or more additional polynucleotides encoding the at least one polypeptide may be modified to remove one or more nucleotide sequences in the corresponding mRNA(s) that are recognizable by the toxin component of said TA module. In some embodiments, the one or more additional polynucleotide(s) encoding the at least one polypeptide may be modified to replace one or more nucleotide sequences in the corresponding mRNA(s) that are recognizable by the toxin component of said TA module, or a functional fragment or derivative thereof, so that such mRNA(s) become resistant to the destruction by said toxin component. In some embodiments, the one or more nucleotide sequence(s) in the corresponding mRNA(s) that are recognizable by the toxin component of that TA module may be Adenine-Cytosine- Adenine (ACA) sequences. In certain embodiments, the ACA sequences in the corresponding mRNA(s) may be replaced. The ACA sequence(s) in the corresponding mRNA(s) may be replaced such that the at least one polypeptide encoded by the one or more additional polynucleotide(s) is the same as when the ACA sequence(s) is not replaced.

[00239] In some embodiments, genetically modified cells disclosed herein may comprise a first polynucleotide encoding a fusion polypeptide comprising a toxin component of a TA module, wherein the toxin component is operably linked to a signal peptide, thereby leading to the secretion of the fusion polypeptide to the space outside of the cytoplasmic membrane of the cell, e.g., periplasm and/or to the extracellular space. Non-limiting examples of a signal peptide are a sec-dependent signal peptide, a signal recognition particle (SRP)-dependent signal peptide, a twin-arginine translocation (TAT)-dependent signal peptide, an HlyA signal peptide.

[00240] In some embodiments, the signal peptide is a Sec-dependent signal peptide. In various embodiments, the Sec-dependent signal peptide may be selected from LamB, LTB, MalE, OmpA, PelB, PhoA, SpA, PhoE, OmpT, OmpF, LivK or functional fragments or derivatives thereof. In some embodiments, the signal peptide may be a PelB signal peptide or functional fragments or derivatives thereof. [00241] In some embodiments, the signal peptide may be a PelB signal peptide. In some embodiments, the PelB signal peptide may comprise the amino acid sequence of MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 1), or a variant thereof. In some embodiments, the PelB signal peptide may comprise the amino acid sequence of MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 1), or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such amino acid sequence.

[00242] In some embodiments, the signal peptide is a signal recognition particle (SRP)- dependent signal peptide. As a non-limiting example, the SRP-dependent signal peptide may be selected from TorT, TolB, DsbA, and functional fragments or derivatives thereof.

[00243] In some embodiments, the signal peptide is a twin-arginine translocation (TAT)- dependent signal peptide. By way of a non-limiting example, the (TAT)-dependent signal peptide may be Tor A, or functional fragments or derivatives thereof. By way of another nonlimiting example, the (TAT)-dependent signal peptide may be Tap, or functional fragments or derivatives thereof.

[00244] In some embodiments, the signal peptide may comprise a twin-arginine translocation TAT-dependent peptide, or functional fragments or derivatives thereof. The TAT-dependent peptide which may be used in accordance with the invention include TorA and Tap, and functional fragments or derivatives thereof.

[00245] In some embodiments, the signal peptide may be a TorA signal peptide or functional fragment or derivative thereof. In some embodiments, the TorA signal peptide may comprise the amino acid sequence of MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 2), or a variant thereof. In some embodiments, the TorA signal peptide may comprise the amino acid sequence of

MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 2) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such amino acid sequence.

[00246] In some embodiments, the signal peptide may be an HlyA signal peptide.

[00247] In certain embodiments, the first polynucleotide and/or the one or more additional polynucleotide sequences described herein are operatively linked to expression (e.g., transcriptional or translational) control sequence(s), e.g., promoters, for example, to direct or modulate nucleic acid synthesis and/or expression. The nucleic acids of the invention can be expressed by any of various appropriate choice of promoters, vectors, media, and the like. The expression control sequence may be in an expression vector.

[00248] In some embodiments, promoters which may be used in accordance with the disclosure may be prokaryotic promoters or eukaryotic promoters. Non-limiting examples of prokaryotic promoters include, e.g., Sp6, araBAD, Ptac, lad, lacZ, T3, T7, T71ac, lac, gpt, lambda PR, PL, Pueto-i and trp. Non-limiting examples of eukaryotic promoters, include, e.g., CMV immediate early, EFla, SV40 (early and late), PGK1 (human or mouse), Ubc, human beta actin, CAG, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, GALI, 10, TEF1, GDS, ADH1, CaMV35S, Virus, Ubi, Hl, U6, LTRs from retrovirus, mouse metallothionein I, and HSV thymidine kinase. As an example, a fungal promotor may include, without limitation, a V factor promoter. Other promoters known by those skilled in the art to control expression of genes in, e.g., prokaryotic or eukaryotic cells or their viruses may also be used.

[00249] A promoter sequence may be operably linked to a coding sequence when RNA polymerase which initiates transcription at the promoter will transcribe the coding sequence into mRNA. Promoters suitable for expressing a polypeptide in bacteria include the E. coli lac or trp promoters, the lad promoter, the lacz promoter, the T3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter, the lambda PL promoter, promoters from operons encoding glycolytic enzymes such as 3 -phosphoglycerate kinase (PGK), and the acid phosphatase promoter. Other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses/bacteriophages may also be used.

[00250] In some embodiments, the expression of the polynucleotides disclosed herein may be under the control of a promoter. In some embodiments, the promotor may be a constitutive promoter. In some embodiments, the promoter may be an inducible promoter.

[00251] In certain embodiments, the polynucleotide sequence encoding any of the fusion polypeptides comprising a metabolic regulator, or a component or functional fragment or derivative thereof, and/or encoding any of the one more additional polypeptides disclosed herein, may further comprise a promoter sequence. In some embodiments, the promoter sequence may be, for example, a constitutive promoter sequence or an inducible promoter sequence for controlling expression of the fusion polypeptide.

[00252] In one aspect, the genetically modified cells of the present disclosure comprise a polynucleotide sequence encoding a fusion polypeptide comprising a toxin component of a TA system, wherein the toxin component is operably linked to a signal peptide, which promotes the secretion of the fusion polypeptide to the periplasm and/or to the extracellular space of a microbial cell expressing the fusion polypeptide. In certain embodiments, the fusion polypeptide is under the control of a promoter such as, but not limited to, a constitutive promoter or an inducible promoter. In certain embodiments, the polynucleotide sequence further comprises a promoter sequence. In some embodiments, the promoter sequences is a constitutive promoter sequence or an inducible promoter sequence for controlling expression of the fusion polypeptide. In one aspect, the invention provides a vector comprising the polynucleotide sequence.

[00253] In some embodiments, the promoter is a constitutive promotor. By way of a nonlimiting example, a constitutive promoter may be a Pbla or PRNAII. Other examples of constitutive promoters may include, without limitation, CMV, EFla, SV40, PGK1 (human or mouse), Ubc, human beta actin, CAG, Ac5, Polyhedrin, TEF1, GDS, CaMV35S, Ubi, Hl, and U6.

[00254] In some embodiments, the promoter is an inducible promoter. Non-limiting examples of an inducible promoter are Hsp70- and Hsp90- derived promoters, lac, sp6, and an T7 promotor. In some embodiments, the inducible promoter is a T7 promoter. In some embodiments, the T7 promoter is a modified T7 promotor. In some embodiments, the modified T7 promoter is selected from an H9 promoter, an G6 promoter, an T448 promoter, an B14 promoter, an B 121 promoter, an B282 promoter, and an B233 promoter.

[00255] In some embodiments, the modified T7 promoter comprises a sequence selected from SEQ ID NOs: 3-9, or variants thereof.

[00256] In some embodiments, polynucleotide sequences described herein comprise a modified T7 promoter sequence comprising a sequence selected from SEQ ID NOs: 3-9, or variants thereof.

[00257] In another aspect, polynucleotide sequences described herein are present in a recombinant construct comprising a modifiedT7 promoter sequence comprising a sequence selected from SEQ ID NOs: 3-9, or variants thereof. In some embodiments, the modified T7 promoter may be a H9 promoter. In some embodiments, the H9 promoter may comprise the nucleotide sequence of TAATACGACTCACTAATACTGAA (SEQ ID NO: 3), or a variant thereof. In some embodiments, the H9 promoter may comprise the nucleotide sequence of TAATACGACTCACTAATACTGAA (SEQ ID NO: 3) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence.

[00258] In some embodiments, the modified T7 promoter may be a G6 promoter. In some embodiments, the G6 promoter may comprise the nucleotide sequence of TAATACGACTCACTATTTCGGAA (SEQ ID NO: 4), or a variant thereof. In some embodiments, the G6 promoter may comprise the nucleotide sequence of TAATACGACTCACTATTTCGGAA (SEQ ID NO: 4) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence.

[00259] In some embodiments, the modified T7 promoter may be a T448 promoter. In some embodiments, the T448 promoter may comprise the nucleotide sequence of TAATACGACTCTCTATAGG (SEQ ID NO: 5), or a variant thereof. In some embodiments, the T448 promoter may comprise the nucleotide sequence of TAATACGACTCTCTATAGG (SEQ ID NO: 5) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence.

[00260] In some embodiments, the modified T7 promoter may be a T448 promoter. In some embodiments, the T448 promoter may comprise the nucleotide sequence of

TAATACGACTCTCTATAGGGAGA (SEQ ID NO: 26), or a variant thereof. In some embodiments, the T448 promoter may comprise the nucleotide sequence of

TAATACGACTCTCTATAGGGAGA (SEQ ID NO: 26) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence.

[00261] In some embodiments, the modified T7 promoter may be a B 14 promoter. In some embodiments, the B14 promoter may comprise the nucleotide sequence of

TAATACGACTCACTATAGGAGAA (SEQ ID NO: 6), or a variant thereof. In some embodiments, the B14 promoter may comprise the nucleotide sequence of

TAATACGACTCACTATAGGAGAA (SEQ ID NO: 6) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence.

[00262] In some embodiments, the modified T7 promoter may be a B 121 promoter. In some embodiments, the B121 promoter may comprise the nucleotide sequence of

TAATACCACTCACTATAGGGAGA (SEQ ID NO: 7), or a variant thereof. In some embodiments, the B121 promoter may comprise the nucleotide sequence of

TAATACCACTCACTATAGGGAGA (SEQ ID NO: 7) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence.

[00263] In some embodiments, the modified T7 promoter may be a B282 promoter. In some embodiments, the B282 promoter may comprise the nucleotide sequence of

TAATACAACTCACTATAGGGAGA (SEQ ID NO: 8), or a variant thereof. In some embodiments, the B282 promoter may comprise the nucleotide sequence of

TAATACAACTCACTATAGGGAGA (SEQ ID NO: 8) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence.

[00264] In some embodiments, the modified T7 promoter may be a B233 promoter. In some embodiments, the B233 promoter may comprise the nucleotide sequence of

TAATACGTCTCACTATAGGGGAA (SEQ ID NO: 9), or a variant thereof. In some embodiments, the B233 promoter may comprise the nucleotide sequence of

TAATACGTCTCACTATAGGGGAA (SEQ ID NO: 9) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,

89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence.

[00265] In some embodiments, the promoter is a T448 promotor comprising the nucleotide sequence TAATACGACTCTCTATAGG (SEQ ID NO: 5).

[00266] In some embodiments, the promoter is a T448 promoter comprising the nucleotide sequence of TAATACGACTCTCTATAGGGAGA (SEQ ID NO: 26).

[00267] In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is a tetracycline (Tc)-inducible promoter. In some embodiments, the inducible promoter is an inducible T7 promotor, for example, without limitation, any of the modified T7 promoter disclosed herein.

[00268] In some embodiments, the inducible promoter may be a tetracycline (Tc)-inducible promoter. In some embodiments, the tetracycline (Tc)-inducible promoter may comprise the nucleotide sequence of 5 -GTTGACACTCTATCATTGATAGAGTTATTTTACCACTC-3 (SEQ ID NO: 69), or a variant thereof. In some embodiments, the tetracycline (Tc)-inducible promoter may comprise the nucleotide sequence of 5 - GTTGACACTCTATCATTGATAGAGTTATTTTACCACTC-3 (SEQ ID NO: 69) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence.

[00269] In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of 1-1500 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)- inducible promoter is induced in the presence of from about 1 ng/ml to at least about 50 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 50 ng/ml to at least about 100 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 150 ng/ml to at least about 200 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 200 ng/ml to at least about 250 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)- inducible promoter is induced in the presence of from about 250 ng/ml to at least about 300 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 300 ng/ml to at least about 350 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 350 ng/ml to at least about 400 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 400 ng/ml to at least about 450 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 450 ng/ml to at least about 500 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 500 ng/ml to at least about 550 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 550 ng/ml to at least about 600 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 600 ng/ml to at least about 650 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 650 ng/ml to at least about 700 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 700 ng/ml to at least about 750 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 750 ng/ml to at least about 800 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 800 ng/ml to at least about 850 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 850 ng/ml to at least about 900 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 900 ng/ml to at least about 950 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 950 ng/ml to at least about 1000 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1000 ng/ml to at least about 1050 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1050 ng/ml to at least about 1100 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1100 ng/ml to at least about 1150 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1150 ng/ml to at least about 1200 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1200 ng/ml to at least about 1250 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1250 ng/ml to at least about 1300 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1300 ng/ml to at least about 1350 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1350 ng/ml to at least about 1400 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1400 ng/ml to at least about 1450 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1450 ng/ml to at least about 1500 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from at least about 100 ng/ml, 120 ng/ml, 140 ng/ml, 160 ng/ml, 180 ng/ml, 200 ng/ml, 220 ng/ml, 240 ng/ml, 260 ng/ml, 280 ng/ml, 300 ng/ml, 320 ng/ml, 340 ng/ml, 360 ng/ml, 380 ng/ml, 400 ng/ml, 420 ng/ml, 440 ng/ml, 460 ng/ml, 480 ng/ml, 500 ng/ml, 520 ng/ml, 540 ng/ml, 560 ng/ml, 580 ng/ml, 600 ng/ml, 620 ng/ml, 640 ng/ml, 660 ng/ml, 680 ng/ml, 700 ng/ml, 720 ng/ml, 740 ng/ml, 760 ng/ml, 780 ng/ml, 800 ng/ml, 820 ng/ml, 840 ng/ml, 860 ng/ml, 880 ng/ml, 900 ng/ml, 920 ng/ml, 940 ng/ml, 960 ng/ml, 980 ng/ml, 1000 ng/ml, 1020 ng/ml, 1040 ng/ml, 1060 ng/ml, 1080 ng/ml, 1100 ng/ml, 1120 ng/ml, 1140 ng/ml, 1160 ng/ml, 1180 ng/ml, 1200 ng/ml, 1220 ng/ml, 1240 ng/ml, 1260 ng/ml, 1280 ng/ml, 1300 ng/ml, 1320 ng/ml, 1340 ng/ml, 1360 ng/ml, 1380 ng/ml, 1400 ng/ml, 1420 ng/ml, 1440 ng/ml, 1460 ng/ml, 1480 ng/ml or 1500 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of about 500 ng/ml anhydrotetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of about 1000 ng/ml anhydrotetracycline.

[00270] In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of 1-1500 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1 ng/ml to at least about 50 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 50 ng/ml to at least about 100 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 150 ng/ml to at least about 200 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 200 ng/ml to at least about 250 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 250 ng/ml to at least about 300 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 300 ng/ml to at least about 350 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)- inducible promoter is induced in the presence of from about 350 ng/ml to at least about 400 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 400 ng/ml to at least about 450 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 450 ng/ml to at least about 500 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)- inducible promoter is induced in the presence of from about 500 ng/ml to at least about 550 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 550 ng/ml to at least about 600 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 600 ng/ml to at least about 650 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)- inducible promoter is induced in the presence of from about 650 ng/ml to at least about 700 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 700 ng/ml to at least about 750 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 750 ng/ml to at least about 800 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)- inducible promoter is induced in the presence of from about 800 ng/ml to at least about 850 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 850 ng/ml to at least about 900 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 900 ng/ml to at least about 950 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)- inducible promoter is induced in the presence of from about 950 ng/ml to at least about 1000 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1000 ng/ml to at least about 1050 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1050 ng/ml to at least about 1100 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1100 ng/ml to at least about 1150 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1150 ng/ml to at least about 1200 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1200 ng/ml to at least about 1250 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1250 ng/ml to at least about 1300 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1300 ng/ml to at least about 1350 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1350 ng/ml to at least about 1400 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1400 ng/ml to at least about 1450 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from about 1450 ng/ml to at least about 1500 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of from at least about 100 ng/ml, 120 ng/ml, 140 ng/ml, 160 ng/ml, 180 ng/ml, 200 ng/ml, 220 ng/ml, 240 ng/ml, 260 ng/ml, 280 ng/ml, 300 ng/ml, 320 ng/ml, 340 ng/ml, 360 ng/ml, 380 ng/ml, 400 ng/ml, 420 ng/ml, 440 ng/ml, 460 ng/ml, 480 ng/ml, 500 ng/ml, 520 ng/ml, 540 ng/ml, 560 ng/ml, 580 ng/ml, 600 ng/ml, 620 ng/ml, 640 ng/ml, 660 ng/ml, 680 ng/ml, 700 ng/ml, 720 ng/ml, 740 ng/ml, 760 ng/ml, 780 ng/ml, 800 ng/ml, 820 ng/ml, 840 ng/ml, 860 ng/ml, 880 ng/ml, 900 ng/ml, 920 ng/ml, 940 ng/ml, 960 ng/ml, 980 ng/ml, 1000 ng/ml, 1020 ng/ml, 1040 ng/ml, 1060 ng/ml, 1080 ng/ml, 1100 ng/ml, 1120 ng/ml, 1140 ng/ml, 1160 ng/ml, 1180 ng/ml, 1200 ng/ml, 1220 ng/ml, 1240 ng/ml, 1260 ng/ml, 1280 ng/ml, 1300 ng/ml, 1320 ng/ml, 1340 ng/ml, 1360 ng/ml, 1380 ng/ml, 1400 ng/ml, 1420 ng/ml, 1440 ng/ml, 1460 ng/ml, 1480 ng/ml or 1500 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of about 500 ng/ml tetracycline. In some embodiments, the tetracycline (Tc)-inducible promoter is induced in the presence of about 1000 ng/ml tetracycline.

[00271] In some embodiments, the Tc-inducible promoter is induced in the presence of anhydrotetracycline or tetracycline at a concentration of about 1 - 200 ng/ml. In some embodiments, the promotor is induced in the presence of anhydrotetracycline or tetracycline at a concentration of about 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 45 ng/ml, 50 ng/ml, 55 ng/ml, 60 ng/ml, 65 ng/ml, 70 ng/ml, 75 ng/ml, 80 ng/ml, 85 ng/ml, 90 ng/ml, 95 ng/ml, 100 ng/ml, 105 ng/ml, 110 ng/ml, 115 ng/ml, 120 ng/ml, 125 ng/ml, 130 ng/ml, 135 ng/ml, 140 ng/ml, 145 ng/ml, 150 ng/ml, 155 ng/ml, 160 ng/ml, 165 ng/ml, 170 ng/ml, 175 ng/ml, 180 ng/ml, 185 ng/ml, 190 ng/ml, 195 ng/ml, or 200 ng/ml. In one aspect, the present disclosure provides a modified T7 promoter that may comprise an nucleotide sequence selected from SEQ ID NOs: 3-9.

[00272] In some embodiments, the modified T7 promoter may comprise the nucleotide sequence of TAATACGACTCACTAATACTGAA (SEQ ID NO: 3), or variant thereof. In some embodiments, the modified T7 promoter may comprise the nucleotide sequence of TAATACGACTCACTATTTCGGAA (SEQ ID NO: 4), or variant thereof. In some embodiments, the modified T7 promoter may comprise the nucleotide sequence of TAATACGACTCTCTATAGG (SEQ ID NO: 5), or variant thereof. In some embodiments, the modified T7 promoter may comprise the nucleotide sequence of TAATACGACTCTCTATAGGGAGA (SEQ ID NO: 26), or variant thereof. In some embodiments, the modified T7 promoter may comprise the nucleotide sequence of TAATACGACTCACTATAGGAGAA (SEQ ID NO: 6), or variant thereof. In some embodiments, the modified T7 promoter may comprise the nucleotide sequence of TAATACCACTCACTATAGGGAGA (SEQ ID NO: 7) or variant thereof. In some embodiments, the T7 promoter may comprise the nucleotide sequence of TAATACAACTCACTATAGGGAGA (SEQ ID NO: 8), or variant thereof. In some embodiments, the modified T7 promoter may comprise the nucleotide sequence of TAATACGTCTCACTATAGGGGAA (SEQ ID NO: 9), or variant thereof.

[00273] In one aspect, the present disclosure provides any of the modified T7 promoters disclosure herein. In another aspects, the present disclosure provides a construct comprising any of the modified T7 promoter sequences disclosed herein.

[00274] In some embodiments of any of the genetically modified cell compositions and/or methods disclosed herein, the first polynucleotide and/or the one or more additional polynucleotides may be integrated into a cellular chromosome. In some embodiments, the first polynucleotide may be integrated into a cellular chromosome into any of various loci. As a non-limiting example, the first polynucleotide may be integrated into a cellular chromosome into the locus of the endogenous toxin and/or antitoxin genes. In some embodiments, the second polynucleotide may be integrated into a cellular chromosome.

[00275] In some embodiments of any of the genetically modified cell compositions and/or methods disclosed herein, the first polynucleotide and/or the one or more additional polynucleotides may be present on a plasmid. Non-limiting examples of plasmids include, e.g., pGBKT7, pISA, pkD46, plnt/Xis, and pASK-IBA4. In some embodiments, the first polynucleotide is present on a plasmid. In some embodiments, the one or more additional polynucleotides is present on a plasmid.

[00276] In one aspect, provided herein is a genetically modified E. coli cell comprising a first polynucleotide encoding a fusion polypeptide comprising MazF toxin operably linked to a signal peptide pelB or TorA, wherein expression of the fusion polypeptide is under the control of a modified T7 promoter or a tetracycline (Tc)-inducible promoter, and wherein the endogenous toxin-antitoxin (TA) module MazEF has been deleted. In some embodiments, the E. coli cell may comprise MazF, wherein the MazF comprises an amino acid sequence MVSRYVPDMGDLIWVDFDPTKGSEQAGHRPAVVLSPFMYNNKTGMCLCVPCTTQS KGYPFEVVLSGQERDGVALADQVKSIAWRARGATKKGTVAPEELQLIKAKINVLIG (SEQ ID NO: 62). the In some embodiments, the PelB signal peptide may comprise the amino acid sequence MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 1). In some embodiments, the TorA signal peptide may comprise the amino acid sequence MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 2). In some embodiments, the modified T7 promoter may comprise a sequence selected from H9 promoter comprising the sequence TAATACGACTCACTAATACTGAA (SEQ ID NO: 3), G6 promoter comprising the sequence TAATACGACTCACTATTTCGGAA (SEQ ID NO: 4), T448 promoter comprising the sequence TAATACGACTCTCTATAGG (SEQ ID NO: 5), T448 promoter comprising the sequence TAATACGACTCTCTATAGGGAGA (SEQ ID NO: 26), B14 promoter comprising the sequence TAATACGACTCACTATAGGAGAA (SEQ ID NO: 6), B121 promoter comprising the sequence TAATACCACTCACTATAGGGAGA (SEQ ID NO: 7), B282 promoter comprising the sequence TAATACAACTCACTATAGGGAGA (SEQ ID NO: 8), and B233 promoter comprising the sequence TAATACGTCTCACTATAGGGGAA (SEQ ID NO: 9). In some embodiments, the modified T7 promoter may be a T448 promoter comprising the sequence TAATACGACTCTCTATAGG (SEQ ID NO: 5). In some embodiments, the modified T7 promoter may be a T448 promoter comprising the sequence TAATACGACTCTCTATAGGGAGA (SEQ ID NO: 26). In some embodiments, the tetracycline (Tc)-inducible promoter may comprise the sequence GTTGACACTCTATCATTGATAGAGTTATTTTACCACTC (SEQ ID NO: 69). In some embodiments, the first polynucleotide may be integrated into a cellular chromosome. In some embodiments of any of the genetically modified E. coli cell disclosed herein, the E. coli cell may further comprise one or more additional polynucleotides. In certain embodiments, the first polynucleotide and/or the additional polynucleotide(s) may encode at least one polypeptide which may comprise a biological substance or which participates in the production of the biological substance, wherein the first polynucleotide encoding the fusion polypeptide and/or the additional polynucleotide encoding the at least one polypeptide is modified to replace one or more ACA nucleotide sequences in the corresponding mRNA(s). The ACA sequence(s) in the corresponding mRNA(s) may be replaced such that the fusion polypeptide encoded by the first polynucleotide and/or the at least one polypeptide encoded by the one or more additional polynucleotide(s) is the same as when the ACA sequence(s) is not replaced. In some embodiments of any of the genetically modified E. coli cell disclosed herein the biological substance may be selected from biomass or its constituents, plasmid DNA, recombinant protein, peptide, amino acid, antibiotic, biosurfactant, biological fuel, organic acid, fatty acid, polyol, flavor, fragrance, polynucleotide, nucleotide, vitamin, pigment, sugar, polysaccharide, biopolymer, and plastic. In one aspect provided herein a method of producing a biological substance, comprising culturing any of the genetically modified cells of the present disclosure under conditions suitable for producing the biological substance, and optionally purifying the biological substance from the cell. As a non-limiting example, the biological substance is biomass or its constituents, and/or produces of biosynthesis localized either intracellularly or extracellularly. Additional examples of a biological substance are, without limitation, a recombinant protein, peptide, amino acid, enzyme, antibiotic, biosurfactant, biological fuel, organic acid, fatty acid, polyol, flavor, fragrance, polynucleotide, nucleotide, vitamin, pigment, sugar, polysaccharide, biopolymer or plastic. In some embodiments, the biological substance may be plasmid DNA. In some embodiments, the biological substance may be localized intracellularly. In some embodiments, the biological substance may be localized extracellularly.

[00277] In one aspect, the present disclosure provides a method of producing a biological substance, the method comprising culturing of any of the genetically modified cells disclosed herein under conditions suitable for producing the biological substance, and optionally purifying the biological substance.

[00278] In some embodiments, a genetically modified cell disclosed herein may express a fusion polypeptide disclosed herein and the expression of the fusion polypeptide in the cell results in an increased efficiency of substrate utilization by the cell as compared to a control cell which does not express the fusion polypeptide. In some embodiments, the increased efficiency of substrate utilization is manifested in an increased production of biomass or a biological substance, or combination thereof, from the same amount of substrate utilized by the cell as compared to the control cell.

[00279] In some embodiments, a genetically modified cell disclosed herein may express a fusion polypeptide disclosed herein and the expression of the fusion polypeptide in the cell results in an increased production of biomass or a biological substance, or combination thereof, as compared to a control cell which does not express the fusion polypeptide.

[00280] In some embodiments, the expression of a fusion polypeptide described herein within a cell described herein can result in a slower rate of substrate utilization by the cell as compared to a control cell which does not express the fusion polypeptide. In some embodiments, the slower rate of substrate utilization is manifested in an increased duration of cellular growth.

[00281] In some embodiments, a fusion polypeptide described herein expressed within a cell described herein possesses an enzymatic activity. In some embodiments, when a fusion polypeptide described herein expressed within a cell described herein possesses an enzymatic activity, the enzymatic activity may be inherent to the fusion polypeptide. In some embodiments, a fusion polypeptide described herein expressed within a cell described herein does not possess an enzymatic activity.

Polynucleotides and Promoters

[00282] In one aspect, the present disclosure provides polynucleotide molecules comprising polynucleotide sequences encoding fusion polypeptides comprising any of the various global metabolic regulators, or components or functional fragments or derivatives thereof, disclosed herein. The global metabolic regulator, or a component or functional fragment or derivative thereof, may be operably linked to a signal peptide which may enable secretion of the fusion polypeptide to the space outside of the cytoplasmic membrane of a genetically modified cell upon expression of the fusion polypeptide in the cell. [00283] In various embodiments, the expression of the fusion polypeptide in the cell may not inhibit the growth of the cell. In various embodiments, the expression of the fusion polypeptide in the cell may not fully inhibit the growth of the cell. In some embodiments, the growth rate (e.g., specific growth rate) of the cell during the expression of the fusion polypeptide may be higher than 0 h’¹. As a non-limiting example, the growth rate may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 h . In certain embodiments, the expression of the fusion polypeptide in the cell may not inhibit the growth of the cell such that the cell is actively dividing. In certain embodiments, the expression of the fusion polypeptide in the cell may not fully inhibit the growth of the cell such that the cell is actively dividing. In some embodiments, the expression of the fusion polypeptide in the cell may not inhibit the growth of the cell such that the cell is proliferating. In some embodiments, the expression of the fusion polypeptide in the cell may not fully inhibit the growth of the cell such that the cell is proliferating.

[00284] In some embodiments, polynucleotide molecules comprising polynucleotide sequences of the present disclosure may encode fusion polypeptides comprising a global metabolic regulator, or a component or functional fragment or derivative thereof. In some embodiments, the global metabolic regulator may be operably linked to a signal peptide. In some embodiments, the global metabolic regulator may comprise a toxin and antitoxin (TA) module, or a component or functional fragment or derivative thereof. In some embodiments, the global metabolic regulator, or a component or functional fragment or derivative thereof, may comprise a toxin component of a TA module.

[00285] In some embodiments, polynucleotide molecules comprising polynucleotide sequences described herein may encode fusion polypeptides comprising a toxin component of a TA module.

[00286] In some embodiments, the fusion polypeptide encoded by the polynucleotide sequences may comprise the same toxin component as the toxin component of an endogenous TA module of the cell and the activity of the endogenous TA module of the cell may be inactivated, for example, by deletion of said endogenous TA module. In various embodiments, the endogenous TA module of the cell may be inactivated by deletion of the gene encoding the endogenous toxin component. In various embodiments, the activity of endogenous TA module of the cell may be inactivated by deletion of the gene encoding the endogenous toxin component, and optionally also by deletion of the gene encoding the corresponding antitoxin component. In some embodiments, the activity of the endogenous TA module of the cell may be inactivated by deletion of the gene encoding the endogenous toxin component and the gene encoding the endogenous antitoxin component. In some embodiments, the TA module may be MazF and MazE (i.e., MazEF), and the toxin component may be MazF, or a functional fragment or derivative thereof. In some embodiments, the TA module may be MazF and MazE (i.e., MazEF), and the antitoxin component may be MazE, or a functional fragment or derivative thereof.

[00287] In one aspect, the invention provides polynucleotide molecules comprising polynucleotide sequences for production of a biological substance expressed in genetically modified cell, e.g., a prokaryotic cell such as, but not limited to, a microbial cell, the cell comprising a polynucleotide molecule comprising a first polynucleotide encoding a fusion polypeptide that may comprise a toxin component of a TA module, e.g., a prokaryotic TA module, wherein the toxin component may be operably linked to a signal peptide which enables the secretion of the fusion polypeptide to a space outside of the cytoplasmic membrane of said cell, e.g., the periplasm and/or to the extracellular space of the cell. In some embodiments, the expression of the fusion polypeptide may be under the control of a constitutive or an inducible promoter. In some embodiments, the polynucleotide molecule comprising the polynucleotide sequence may comprise one or more additional polynucleotide sequences encoding at least one polypeptide which is a biological substance or which participates in the production of said biological substance. In some embodiments, the one or more additional polynucleotide(s) may be modified to remove one or more nucleotide sequences in the corresponding mRNA(s) that are recognizable by the toxin component, or fragment or derivative thereof, of said TA module so that such mRNA(s) become resistant to the destruction by the toxin component. In some embodiments, the endogenous TA module in the cells is deleted. In some embodiments, the endogenous TA module in the cells is eliminated.

[00288] In some embodiments, the global metabolic regulator may comprise a TA module. By way of a non-limiting example, the TA module may comprise a microbial TA module, including, without limitation, a bacterial TA module, e.g., an E. coll TA module such as, but not limited to MazEF. In some embodiments, the TA module may comprise MazF and MazE, and the toxin component is MazF, or a functional fragment or derivative thereof. In some embodiments, the global metabolic regulator may be a TA module, and the TA module is MazE, or a functional fragment or derivative thereof, and MazF, or a functional fragment or derivative thereof.

[00289] In various embodiments, the TA module is MazF and MazE (MazEF) and the toxin component is MazF, or a functional fragment or derivative thereof. In some embodiments, MazF, or a functional fragment or derivative thereof, may comprise the amino acid sequence of

MVSRYVPDMGDLIWVDFDPTKGSEQAGHRPAVVLSPFMYNNKTGMCLCVPCTTQS KGYPFEVVLSGQERDGVALADQVKSIAWRARGATKKGTVAPEELQLIKAKINVLIG (SEQ ID NO: 62), or a variant thereof. In some embodiments, the nucleotide sequence encoding the MazF, or a functional fragment or derivative thereof, may comprise the nucleotide sequence set forth in SEQ ID NO: 63. In certain embodiments, the nucleotide sequence encoding the MazF, or functional fragment or derivative thereof, may comprise the nucleotide sequence set forth in SEQ ID NO: 64.

[00290] In various embodiments, the TA module is MazF and MazE (MazEF) and the antitoxin component is MazE, or a functional fragment or derivative thereof. In some embodiments, MazE, or a functional fragment or derivative thereof, may comprise the amino acid sequence of (SEQ ID NO: 65), or a variant thereof. In some embodiments, the nucleotide sequence encoding the MazE, or a functional fragment or derivative thereof, may comprise the nucleotide sequence set forth in SEQ ID NO: 66.

[00291] In some embodiments, the TA module may comprise MazF, or a functional fragment or derivative thereof, and MazE, or a functional fragment or derivative thereof, and the toxin component may be MazF, or a functional fragment or derivative thereof. In some embodiments, the first polynucleotide may be integrated into the chromosome at any locus, such as but not limited to, the locus of an endogenous toxin and/or antitoxin genes.

[00292] In certain embodiments, when any of the global metabolic regulator(s) operably linked to a signal peptide may be a toxin component of a TA module (e.g., MazEF) such as, but not limited to, MazF, or a functional fragment or derivative thereof, the one or more additional polynucleotide(s) encoding the at least one polypeptide may be modified to replace one or more nucleotide sequences in the corresponding mRNA(s) that are recognizable by said toxin component. In some embodiments, the additional polynucleotide(s) encoding the at least one polypeptide may be modified to replace the one or more nucleotide sequence(s) in the corresponding mRNA(s) that may be recognizable by the toxin with a sequence that is not recognizable (i.e., a non-recognizable sequence) by the toxin sequence. In some embodiments, the nucleotide sequences recognizable by the toxin component of the TA module may comprise the sequence Ad enine-Cytosine- Adenine (AC A). The AC A sequence(s) in the corresponding mRNA(s) may be replaced such that the at least one polypeptide encoded by the one or more additional polynucleotide(s) is the same as when the ACA sequence(s) is not replaced.

[00293] In some embodiments, the global metabolic regulator is a toxin component of a TA module, such as, but not limited to, MazE and MazF. In various embodiments, the TA module is MazF and MazE, and the toxin component is MazF, or a functional fragment or derivative thereof. In various embodiments, when the TA module is MazF and MazE, and the toxin component is MazF, or a functional fragment or derivative thereof, any of the one or more additional polynucleotides encoding the at least one polypeptide may be modified to remove one or more nucleotide sequences in the corresponding mRNA(s) that are recognizable by the MazF, or functional fragment or derivative thereof. In some embodiments, the nucleotide sequences recognizable by the MazF, or functional fragment or derivative thereof, may comprise the sequence Adenine-Cytosine-Adenine (ACA). The ACA sequence(s) in the corresponding mRNA(s) may be replaced such that the at least one polypeptide encoded by the one or more additional polynucleotide(s) is the same as when the ACA sequence(s) is not replaced.

[00294] In some embodiments, polynucleotide molecules comprising polynucleotide sequences encoding any of various polypeptides disclosed herein, e.g., fusion polypeptides comprising a global metabolic regulator, may be expressed in a genetically modified cell of the present disclosure. The genetically modified cells may be any of various eukaryotic cells described herein such as, but not limited to, algae, fungi, and protozoa. In some embodiments, the eukaryotic cell may be a plant cell. In some embodiments, the eukaryotic cell may be an animal cell, e.g., a mammalian cell. In various embodiments, the genetically modified cell of the present disclosure may be any of various prokaryotic cells described herein such as, but not limited to bacterial cells including eubacteria and archaea, e.g., archaebacterial cells. In some embodiments, the genetically modified cell may be a microbial cell (e.g., E. colt).

[00295] In some embodiments, when the genetically modified cell of the present disclosure is a microbial cell, any of the signal peptides disclosed herein may enable the secretion of the fusion polypeptide to the periplasm of the microbial cell. In some embodiments, when the genetically modified cell of the present disclosure is a microbial cell, any of the signal peptides disclosed herein may enable the secretion of the fusion polypeptide to the extracellular space of the microbial cell.

[00296] In some embodiments, polynucleotide molecules comprising polynucleotide sequences disclosed herein may encode any of various polypeptides which may comprise a biological substance(s) or which may participate in the production of such substance(s). As a non-limiting example, a biological substance may be biomass or its constituents, and/or products of biosynthesis localized either intracellularly or extracellularly. In some embodiments, the biological substance may be plasmid DNA. Additional examples of a biological substance, without limitation, are a recombinant protein, peptide, amino acid, enzyme antibiotic, biosurfactant, biological fuel, organic acid, fatty acid, polyol, flavor, fragrance, polynucleotide, nucleotide, vitamin, pigment, sugar, polysaccharide, biopolymer and plastic. In some embodiments, the biological substance is localized intracellularly. In some embodiments, the biological substance is localized extracellularly, for example, in the extracellular space. In some embodiments, the biological substance is localized intracellularly. In some embodiments, the biological substance is localized in the cytoplasm. In some embodiments, the biological substance is localized in the periplasmic space. In some embodiments, the one or more additional polynucleotides encoding the at least one polypeptide may be modified to remove one or more nucleotide sequences in the corresponding mRNA(s) that are recognizable by the toxin component of said TA module. In some embodiments, the one or more additional polynucleotides encoding the at least one polypeptide may be modified to replace one or more nucleotide sequences in the corresponding mRNA(s) that are recognizable by the toxin component of said TA module. In some embodiments, the one or more nucleotide sequence(s) in the corresponding mRNA(s) that are recognizable by the toxin component of that TA module may be Adenine-Cytosine-Adenine (ACA) sequences. In certain embodiments, the ACA sequences in the corresponding mRNA(s) may be replaced by an alternate triplet codon such that the encoded amino acid remains unchanged. In some embodiments, the ACA sequences may be replaced. The ACA sequence(s) in the corresponding mRNA(s) may be replaced such that the at least one polypeptide encoded by the one or more additional polynucleotide(s) is the same as when the ACA sequence(s) is not replaced.

[00297] In some embodiments, polynucleotide molecules comprising polynucleotide sequences encoding fusion polypeptides may comprise a toxin component of a TA module, or a functional fragment or derivative thereof, wherein the toxin component is operably linked to a signal peptide, thereby leading to the secretion of the fusion polypeptide to the space outside of the cytoplasmic membrane of a cell, e.g., periplasm and/or to the extracellular space. Nonlimiting examples of a signal peptide are a sec-dependent signal peptide, a signal recognition particle (SRP)-dependent signal peptide, a twin-arginine translocation (TAT)-dependent signal peptide, an HlyA signal peptide.

[00298] In some embodiments, the signal peptide is a Sec-dependent signal peptide. In various embodiments, the Sec-dependent signal peptide may be selected from LamB, LTB, MalE, OmpA, PelB, PhoA, SpA, PhoE, OmpT, OmpF, LivK or functional fragments or derivatives thereof. In some embodiments, the signal peptide may be a PelB signal peptide or functional fragments or derivatives thereof. [00299] In some embodiments, the signal peptide may be a PelB signal peptide, or a functional fragment or derivative thereof. In some embodiments, the PelB signal peptide may comprise the amino acid sequence of MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 1), or a variant thereof. In some embodiments, the PelB signal peptide may comprise the amino acid sequence of MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 1), or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such amino acid sequence.

[00300] In some embodiments, the signal peptide is a signal recognition particle (SRP)- dependent signal peptide. As a non-limiting example, the SRP-dependent signal peptide may be selected from TorT, TolB, DsbA, and functional fragments or derivatives thereof.

[00301] In some embodiments, the signal peptide is a twin-arginine translocation (TAT)- dependent signal peptide. By way of a non-limiting example, the (TAT)-dependent signal peptide may be TorA, or functional fragments or derivatives thereof. By way of another nonlimiting example, the (TAT)-dependent signal peptide may be Tap, or functional fragments or derivatives thereof.

[00302] In some embodiments, the signal peptide may comprise a twin-arginine translocation TAT-dependent peptide, or functional fragments or derivatives thereof. The TAT-dependent peptide which may be used in accordance with the invention include TorA and Tap, and functional fragments or derivatives thereof.

[00303] In some embodiments, the signal peptide may be a TorA signal peptide or a functional fragment or derivative thereof. In some embodiments, the TorA signal peptide may comprise the amino acid sequence of

MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 2), or a variant thereof. In some embodiments, the TorA signal peptide may comprise the amino acid sequence of MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 2) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such amino acid sequence.

[00304] In some embodiments, the signal peptide may be an HlyA signal peptide.

[00305] In certain embodiments, the disclosure provides nucleic acid (e.g., DNA) sequences operatively linked to expression (e.g., transcriptional or translational) control sequence(s), e.g., promoters, for example, to direct or modulate nucleic acid synthesis and/or expression. [00306] In some embodiments, promoters which may be used in accordance with the disclosure may be any of various prokaryotic promoters and/or eukaryotic promoters disclosed herein.

[00307] In some embodiments, the expression of the polynucleotides disclosed herein may be under the control of a promoter. In some embodiments, the promotor may be a constitutive promoter. In some embodiments, the promoter may be an inducible promoter.

[00308] In certain embodiments, a polynucleotide molecule comprising a polynucleotide sequence encoding any of the fusion polypeptides comprising a metabolic regulator, or a component or functional fragment or derivative thereof, and/or encoding any of the one more additional polypeptides disclosed herein, may further comprise a promoter sequence. In some embodiments, the promoter sequence may be, for example, a constitutive promoter sequence or an inducible promoter sequence for controlling expression of the fusion polypeptide.

[00309] In some embodiments, the promoter is a constitutive promotor.

[00310] In some embodiments, the promoter is an inducible promoter.

[00311] In some embodiments, the modified T7 promoter comprises a sequence selected from SEQ ID NOs: 3-9, or variants thereof.

[00312] In one aspect, the present invention provides a modified T7 promoter sequence comprising a sequence selected from SEQ ID NOs: 3-9, or variants thereof.

[00313] In another aspect, the present invention provides a recombinant construct comprising a modified T7 promoter sequence comprising a sequence selected from SEQ ID NOs: 3-9, or variants thereof. In some embodiments, the modified T7 promoter may be a H9 promoter. In some embodiments, the H9 promoter may comprise the nucleotide sequence of TAATACGACTCACTAATACTGAA (SEQ ID NO: 3), or a variant thereof. In some embodiments, the H9 promoter may comprise the nucleotide sequence of TAATACGACTCACTAATACTGAA (SEQ ID NO: 3) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence.

[00314] In some embodiments, the modified T7 promoter may be a G6 promoter. In some embodiments, the G6 promoter may comprise the nucleotide sequence of

TAATACGACTCACTATTTCGGAA (SEQ ID NO: 4), or a variant thereof. In some embodiments, the G6 promoter may comprise the nucleotide sequence of

TAATACGACTCACTATTTCGGAA (SEQ ID NO: 4) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence.

[00315] In some embodiments, the modified T7 promoter may be a T448 promoter. In some embodiments, the T448 promoter may comprise the nucleotide sequence of TAATACGACTCTCTATAGG (SEQ ID NO: 5), or a variant thereof. In some embodiments, the T448 promoter may comprise the nucleotide sequence of TAATACGACTCTCTATAGG (SEQ ID NO: 5) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence.

[00316] In some embodiments, the modified T7 promoter may be a T448 promoter. In some embodiments, the T448 promoter may comprise the nucleotide sequence of

TAATACGACTCTCTATAGGGAGA (SEQ ID NO: 26), or a variant thereof. In some embodiments, the T448 promoter may comprise the nucleotide sequence of

TAATACGACTCTCTATAGGGAGA (SEQ ID NO: 26) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence.

[00317] In some embodiments, the modified T7 promoter may be a B 14 promoter. In some embodiments, the B14 promoter may comprise the nucleotide sequence of

TAATACGACTCACTATAGGAGAA (SEQ ID NO: 6), or a variant thereof. In some embodiments, the B14 promoter may comprise the nucleotide sequence of

TAATACGACTCACTATAGGAGAA (SEQ ID NO: 6) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence.

[00318] In some embodiments, the modified T7 promoter may be a B 121 promoter. In some embodiments, the B121 promoter may comprise the nucleotide sequence of TAATACCACTCACTATAGGGAGA (SEQ ID NO: 7), or a variant thereof. In some embodiments, the B121 promoter may comprise the nucleotide sequence of TAATACCACTCACTATAGGGAGA (SEQ ID NO: 7) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence. [00319] In some embodiments, the modified T7 promoter may be a B282 promoter. In some embodiments, the B282 promoter may comprise the nucleotide sequence of

TAATACAACTCACTATAGGGAGA (SEQ ID NO: 8), or a variant thereof. In some embodiments, the B282 promoter may comprise the nucleotide sequence of

TAATACAACTCACTATAGGGAGA (SEQ ID NO: 8) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence.

[00320] In some embodiments, the modified T7 promoter may be a B233 promoter. In some embodiments, the B233 promoter may comprise the nucleotide sequence of TAATACGTCTCACTATAGGGGAA (SEQ ID NO: 9), or a variant thereof. In some embodiments, the B233 promoter may comprise the nucleotide sequence of TAATACGTCTCACTATAGGGGAA (SEQ ID NO: 9) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence.

[00321] In some embodiments, the promoter is a T448 promotor comprising the sequence TAATACGACTCTCTATAGG (SEQ ID NO: 5).

[00322] In some embodiments, the promoter is a T448 promotor comprising the sequence TAATACGACTCTCTATAGGGAGA (SEQ ID NO: 26).

[00323] In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is a tetracycline (Tc)-inducible promoter.

[00324] In some embodiments, the inducible promoter may be a tetracycline (Tc)-inducible promoter. In some embodiments, the tetracycline (Tc)-inducible promoter may comprise the nucleotide sequence of GTTGACACTCTATCATTGATAGAGTTATTTTACCACTC (SEQ ID NO: 69), or a variant thereof. In some embodiments, the tetracycline (Tc)-inducible promoter may comprise the nucleotide sequence of GTTGACACTCTATCATTGATAGAGTTATTTTACCACTC (SEQ ID NO: 69) or a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such nucleotide sequence.

[00325] In some embodiments, the inducible promoter is an isopropyl [3-d-l- thiogalactopyranoside (IPTG)-inducible promoter. As an example, without limitation, the IPTG-inducible promoter can be a T7 promotor or a modified T7 promotor disclosed herein. In some embodiments, the IPTG-inducible promoter may be induced, e.g., by addition of IPTG at a concentration of about at least about 0.000001 mM, at least about 0.00001 mM, at least about 0.0001 mM, at least about 0.001 mM, at least about 0.01 mM, at least about 0.1 mM, at least about 1.0 mM, at least about 1.1 mM, at least about 1.2 mM, at least about 1.3 mM, at least about 1.4 mM, at least about 1.5 mM, at least about 1.6 mM, at least about 1.7 mM, at least about 1.8 mM, at least about 1.9 mM, at least about 2.0 mM, at least about 2.1 mM, at least about 2.2 mM, at least about 2.3 mM, at least about 2.4 mM, at least about 2.5 mM, at least about 2.6 mM, at least about 2.7 mM, at least about 2.8 mM, at least about 2.9 mM, at least about 3.0 mM, at least about 3.1 mM, at least about 3.2 mM, at least about 3.3 mM, at least about 3.4 mM, at least about 3.5 mM, at least about 3.6 mM, at least about 3.7 mM, at least about 3.8 mM, at least about 3.9 mM, at least about 4.0 mM, at least about 4.1 mM, at least about 4.2 mM, at least about 4.3 mM, at least about 4.4 mM, at least about 4.5 mM, at least about 4.6 mM, at least about 4.7 mM, at least about 4.8 mM, at least about 4.9 mM, or at least about 5.0 mM. In some embodiments, the IPTG-inducible promoter may be induced by addition of IPTG at a concentration of from about 0.000001 mM to about 3.0 mM, from about 0.00001 mM to about 3.0 mM, from about 0.0001 mM to about 3.0 mM, from about 0.001 mM to about 3.0 mM, from about 0.01 mM to about 3.0 mM, or from about 0.1 mM to about 3.0 mM. In some embodiments, the IPTG-inducible promoter may be induced by addition of IPTG at a concentration from about 0.001 mM to about 3.0 mM. In some embodiments, the IPTG- inducible promoter may be induced by addition of IPTG at a concentration from about 0.01 mM to about 3.0 mM. In some embodiments, the IPTG-inducible promoter may be induced by addition of IPTG at a concentration of from about 0.000001 mM to about 2.0 mM, from about 0.00001 mM to about 2.0 mM, from about 0.0001 mM to about 2.0 mM, from about 0.001 mM to about 2.0 mM, from about 0.01 mM to about 2.0 mM, or from about 0.1 mM to about 2.0 mM. In some embodiments, the IPTG-inducible promoter may be induced by addition of IPTG at a concentration from about 0.001 mM to about 2.0 mM. In some embodiments, the IPTG-inducible promoter may be induced by addition of IPTG at a concentration from about 0.01 mM to about 2.0 mM. In some embodiments, the IPTG- inducible promoter may be induced by addition of IPTG at a concentration of from about 0.000001 mM to about 1.0 mM, from about 0.00001 mM to about 1.0 mM, from about 0.0001 mM to about 1.0 mM, from about 0.001 mM to about 1.0 mM, from about 0.01 mM to about 1.0 mM, or from about 0.1 mM to about 1.0 mM. In some embodiments, the IPTG-inducible promoter may be induced by addition of IPTG at a concentration from about 0.001 mM to about 1.0 mM. In some embodiments, the IPTG-inducible promoter may be induced by addition of IPTG at a concentration from about 0.01 mM to about 1.0 mM.

[00326] In one aspect, the present disclosure provides a modified T7 promoter that may comprise a nucleotide sequence selected from SEQ ID NOs: 3-9. In some embodiments, the modified T7 promoter may comprise the nucleotide sequence of TAATACGACTCACTAATACTGAA (SEQ ID NO: 3), or variant thereof. In some embodiments, the modified T7 promoter may comprise the nucleotide sequence of TAATACGACTCACTATTTCGGAA (SEQ ID NO: 4), or variant thereof. In some embodiments, the modified T7 promoter may comprise the nucleotide sequence of TAATACGACTCTCTATAGG (SEQ ID NO: 5), or variant thereof. In some embodiments, the modified T7 promoter may comprise the nucleotide sequence of TAATACGACTCTCTATAGGGAGA (SEQ ID NO: 26), or variant thereof. In some embodiments, the modified T7 promoter may comprise the nucleotide sequence of TAATACGACTCACTATAGGAGAA (SEQ ID NO: 6), or variant thereof. In some embodiments, the modified T7 promoter may comprise the nucleotide sequence of TAATACCACTCACTATAGGGAGA (SEQ ID NO: 7) or variant thereof. In some embodiments, the T7 promoter may comprise the nucleotide sequence of TAATACAACTCACTATAGGGAGA (SEQ ID NO: 8), or variant thereof. In some embodiments, the modified T7 promoter may comprise the nucleotide sequence of TAATACGTCTCACTATAGGGGAA (SEQ ID NO: 9), or variant thereof.

[00327] In some embodiments of any of polynucleotide molecules comprising polynucleotides encoding, e.g., fusion polypeptides, and/or any additional polynucleotides encoding, e.g., a biological substance disclosed herein, may be integrated into a cellular chromosome. In some embodiments, the polynucleotide may be integrated into a cellular chromosome into any of various loci. As a non-limiting example, the polynucleotide may be integrated into a cellular chromosome into the locus of the endogenous toxin and/or antitoxin genes.

[00328] In some embodiments, any of the polynucleotides disclosed herein may be present on a plasmid.

[00329] In one aspect, the present disclosure provides a construct comprising any of the polynucleotide sequences disclosed herein. In some embodiments, the polynucleotide sequence may encode a fusion polypeptide comprising a toxin component of a toxin and antitoxin (TA) module, or a functional fragment or derivative thereof, wherein the toxin component is operably linked to a signal peptide which enables secretion of the fusion polypeptide to the periplasm and/or to the extracellular space of a cell, e.g., a microbial cell disclosed herein, expressing the fusion polypeptide, and wherein the polynucleotide sequence optionally further comprises a constitutive promoter sequence or an inducible promoter sequence for controlling expression of the fusion polypeptide.

[00330] In one embodiment, the present disclosure provides a polynucleotide molecule comprising a polynucleotide sequence encoding a fusion polypeptide comprising MazF toxin operably linked to a signal peptide pelB or Tor A, wherein expression of the polynucleotide sequence optionally further comprises a modified T7 promoter or a tetracycline (Tc)-inducible promoter.

[00331] In some embodiments, a polynucleotide molecule comprising a polynucleotide sequence described herein can encode a fusion polypeptide described herein, and expression of the fusion polypeptide in a cell described herein results in an increased efficiency of substrate utilization by the cell as compared to a control cell which does not express the fusion polypeptide. In some embodiments, the increased efficiency of substrate utilization is manifested in an increased production of biomass or a biological substance, or combination thereof, from the same amount of substrate utilized by the cell as compared to the control cell. [00332] In some embodiments, a polynucleotide molecule comprising a polynucleotide sequence described herein can encode a fusion polypeptide described herein, and expression of the fusion polypeptide in a cell described herein results in an increased production of biomass or a biological substance, or combination thereof, as compared to a control cell which does not express the fusion polypeptide.

[00333] In some embodiments, the expression of the fusion polypeptide in the cell results in a slower rate of substrate utilization by the cell as compared to a control cell which does not express the fusion polypeptide. In some embodiments, the slower rate of substrate utilization is manifested in an increased duration of cellular growth.

[00334] In some embodiments, a polynucleotide molecule comprising a polynucleotide sequence described herein can encode a fusion polypeptide described herein, and the fusion polypeptide possesses an enzymatic activity. In some embodiments, when polynucleotide molecule comprising a polynucleotide sequence described herein encodes a fusion polypeptide described herein which possesses an enzymatic activity, the enzymatic activity may be inherent to the fusion polypeptide. In some embodiments, a polynucleotide molecule comprising a polynucleotide sequence described herein can encode a fusion polypeptide described herein, and the fusion polypeptide does not possess an enzymatic activity. [00335] In one aspect provided herein is a method of increasing efficacy of substrate utilization by a cell described herein, and the method can comprise genetically modifying the cell by introducing a polynucleotide molecule comprising a polynucleotide sequence described herein or a recombinant construct described herein into the cell.

[00336] In another aspect provided herein is a method of increasing production of biomass or a biological substance, or a combination thereof, by a cell described herein, and the method can comprise genetically modifying the cell by introducing a polynucleotide molecule comprising a polynucleotide sequence described herein or a recombinant construct described herein into the cell.

Expression Vectors and Cloning

[00337] In one aspect, the present invention provides expression vectors comprising any of the nucleic acids (e.g., polynucleotides, promoters) of the invention.

[00338] The nucleic acids useful for methods of the invention can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983). J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380: Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; U.S. Pat. No. 4,458,066.

[00339] Techniques for the manipulation of nucleic acids, such as, e.g., subcloning, sequencing, hybridization and the like are well described in the Scientific and patent literature, see, e.g., Sambrook, ed., Molecular Cloning: A Laboratory Manual (2^nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Current Protocols in Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization with Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

[00340] Expression vectors may comprise, without limitation, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for host cells of interest, e.g., microbial hosts in the disclosure.

[00341] In some embodiments, vectors may include chromosomal, non-chromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the art and are commercially available. [00342] Low copy number or high copy number vectors may be employed with the present invention. The expression vector may comprise a promoter, a ribosome binding site for translation initiation and/or a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

[00343] In some embodiments, the expression vectors may comprise one or more selectable marker genes to permit selection of host cells containing the vector. Such selectable markers include, without limitation, genes conferring e.g., tetracycline or ampicillin resistance in E. coli. and the S. cerevisiae TRP1 gene.

[00344] In some embodiments, the vector may be in the form of a plasmid or a phage. A variety of cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by, e.g., Ausubel et al. Current Protocols in Molecular Biology, John Wiley 503 Sons, Inc. 1997 and Sambrook, ed., Molecular Cloning: A Laboratory Manual (2^nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989).

[00345] Particular bacterial vectors which may be used include the commercially available plasmids comprising genetic elements of the well-known cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pOE70, pGE60, pGE-9 (Qiagen), pIDlO, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, DR540, pRIT5 (Pharmacia), pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However, any other vector may be used as long as it is replicable and viable in the host cell.

[00346] Expression vectors of the invention may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed, e.g., genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways. The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is ligated to the desired position in the vector following digestion of the insert and the vector with appropriate restriction endonucleases. Alternatively, blunt ends in both the insert and the vector may be ligated. A variety of cloning techniques are disclosed in Ausubel et al. Current Protocols in Molecular Biology, John Wiley 503 Sons, Inc. 1997 and Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press (1989). Fermentation Processes

[00347] In one aspect, the compositions and methods of the present invention are used in fermentation processes for the production of any of various biological substances disclosed herein. In certain embodiments, a biological substance may encompass biomass or its constituents, and/or products of biosynthesis localized either intracellularly or extracellularly. As a non-limiting example, the biological substance may be, e.g., a recombinant protein, peptide, amino acid, enzyme, antibiotic, biosurfactant, biological fuel, organic acid, fatty acid, polyol, flavor, fragrance, polynucleotide, nucleotide, vitamin, pigment, sugar, polysaccharide, biopolymer, or plastic. As another non-limiting example, the biological substance may be plasmid DNA.

[00348] In some embodiments, the compositions and methods of the present invention are used in fermentation processes for the production of biopharmaceuticals such as, but not limited to, erythropoietin, insulin, blood clotting factor, interferons, human growth hormone, somatotropin, tissue plasminogen activator, interleukin, hirudin, anti-hemophilia factor, parathyroid hormone (e.g., human parathyroid hormone, hPTH), epidermal growth factor and other growth factors, therapeutic monoclonal antibodies, and various therapeutic vaccines.

[00349] In some embodiments, the compositions and methods of the present invention are used in fermentation processes for the production of enzymes such as, but not limited to, chymosin, trypsin, aspartic proteinase, serine proteases, alkaline proteases, esterases, chitinases, tannase, nitrile hydratase, streptokinase, levansucrases, xylanases, cellulases, glucoamylase, alkaline amylases, lipases, pectinases, a-amylase, pullulanase, glucose isomerase, pectate lyase, mannanase, P-glucanase, and keratinase.

[00350] In some embodiments, the compositions and methods of the present invention are used in fermentation processes for the production of antibiotics such as, but not limited to, actinomycin, bleomycin, rifamycin, chloramphenicol, tetracycline, lincomycin, erythromycin, streptomycin, cyclohexamide, puromycin, cycloserine, bacitracin, penicillin, cephalosporin, sancomycin, polymyxin, and gramicidin.

[00351] In some embodiments, the compositions and methods of the present invention are used in fermentation processes for the production of biosurfactants such as, but not limited to, rhamnolipids, sophorolipids, glycolipids, and lipopeptides.

[00352] In some embodiments, the compositions and methods of the present invention are used in fermentation processes for the production of biological fuels such as, but not limited to, bioethanol and biobutanol. [00353] In some embodiments, the compositions and methods of the present invention are used in fermentation processes for the production of amino acids such as, but not limited to, L- glutamate, L-lysine, L-phenylalanine, L-aspartic acid, L-isoleucine, L-Valine, L-tryptophan, L-proline (hydroxyproline), L-threonine, L-methionine, and D-p-hydroxyphenylglycine.

[00354] In some embodiments, the compositions and methods of the present invention are used in fermentation processes for the production of organic acids such as, but not limited to, citric acid, lactic acid, gluconic acid, acetic acid, propionic acid, succinic acid, fumaric acid, and itaconic acid.

[00355] In some embodiments, the compositions and methods of the present invention are used in fermentation processes for the production of fatty acids such as, but not limited to, arachidonic acid, polyunsaturated fatty acid (PUB A), and y-linoleic acid.

[00356] In some embodiments, the compositions and methods of the present invention are used in fermentation processes for the production of polyols such as, but not limited to, glycerol, mannitol, erythritol, and xylitol.

[00357] In some embodiments, the compositions and methods of the present invention are used in fermentation processes for the production of flavors and fragrances such as, but not limited to, vanillin, benzaldehyde, dixydroxyacetone, 4-(R)-decanolide, and 2-actyl-l- pyrroline.

[00358] In some embodiments, the compositions and methods of the present invention are used in fermentation processes for the production of nucleotides such as, but not limited to, 5'- guanylic acid and 5'-inosinic acid.

[00359] In some embodiments, the compositions and methods of the present invention are used in fermentation processes for the production of vitamins such as, but not limited to, vitamin C. vitamin F, vitamin B2, provitamin D2, vitamin B 12, folic acid, nicotinamide, biotin, 2-keto-L-gulonic acid, and provitamin Q10.

[00360] In some embodiments, the compositions and methods of the present invention are used in fermentation processes for the production of pigments such as, but not limited to, astaxathin, P-carotene, leucopene, monascorubrin, and rubropunctatin.

[00361] In some embodiments, the compositions and methods of the present invention are used in fermentation processes for the production of sugars and polysaccharides such as, but not limited to, ribose, sorbose, xanthan, gellan, and dextran.

[00362] In some embodiments, the compositions and methods of the present invention are used in fermentation processes for the production of biopolymers and plastics such as, but not limited to, polyhydroxyalkanoates (PHA), poly-y-glutamic acid, and 1,3-propanediol. EXAMPLES

[00363] The present invention is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.

Materials and Methods

[00364] E. coli T7 Express Iq. E. coli T7 Express Iq (New England Biolabs) are enhanced E. coli BL21 derivative, chemically competent E. coli cells suitable for high efficiency transformation and protein expression. E. coli T7 Express Iq genotype is

MiniF /ac/⁹(Cam^R) / fhuA2 lacZ::T7 genel [Ion] ompT gal sulAll R(mcr-73 : :miniTnlO— Tet^s 2 [dem] R(zgb-210::Tnl0—' Q ) endAl A(mcrC-mrr) 114::IS10. and structural and/or functional features may comprise:

T7 RNA Polymerase in the lac operon - no lambda prophage;

Tight control of expression by laclq allows potentially toxic genes to be cloned;

Deficient in proteases Lon and OmpT;

Resistant to phage T1 (fhuA2), Cam, Nit;

Does not restrict methylated DNA (McrA-, McrBC-, EcoBr-, Mrr-).

[00365] Modifications of T7 promoter. The fragment with a complete MazF nucleotide sequence (SEQ ID Nos: 63-64) was amplified from the chromosome of E. coli BL21 (DE3) using primers 5’-CAT ATG GTA AGC CGA TAC GTA CC-3 ’ (SEQ ID NO: 70) and 5’-AAG CTT CTA CCC AAT CAG TAC GTT AA-3 ’ (SEQ ID NO: 33). The fragment was cloned into NdEl-Hind III sites of pet22b vector (Novagen, UK), containing T7 expression region with T7 promotor. The ligase mixture was transformed into E. coli XLlBlue strain to obtain plasmid pet22b-T7mazF. The sequence was confirmed by sequencing.

[00366] The inducible promoters of different strength upstream of MazF were made by mutant-specific PCR based on the sequence of native T7 promotor (see Table 1) in the mutant strain E. coli T7 Express Iq AmazEF with operon mazEF deleted from the chromosome as described below. E. coli T7 Express Iq contains T7 RNA polymerase in its lac-operon, which allowed use of the native T7 promoter, as well as mutant promoters built of its consensus sequence for target protein expression.

[00367] The native T7 promoter contains two conserved motifs surrounded by variable regions:

TAATACGACTCACTATAGGGGAA (SEQ ID NO: 10) variable motif 1 variable motif 2

[00368] Introduction of mutations in these two conserved motifs enables weakening of promoter efficiency, thereby substantially lowering MazF expression in the cells. Sequences of mutant promoters are shown in Table 1. Mutations were introduced by mutant-specific PCR using the pet22a-T7MazF template. Each primer pair consisted of a direct primer with the necessary changes are shown in Table 2, and a single T7 reverse primer. Each PCR product was separated by 1% agarose electrophoresis, followed by incision of the 550 bp product with MazF under the control of a mutant T7 promoter. DNA was extracted from the gel with SiCh, followed by ligation into pet22b viaBglll/Hindlll restriction sites. The ligase mix was transformed into competent cells A. coli XLlBlue by CaCE transformation method and positive clones were selected. In total, 10 expression constructions with mutant T7 promoters were produced and their validity confirmed by sequencing.

Table 1. Modified T7 promoters, the introduced mutations are highlighted.

Table 2. Nucleotide sequences of direct primers for mutant-specific PCR for plasmid constructions for MazF expression

[00369] dsDNA construct design for MazF secretion into periplasm (pelB signal peptide SEQ ID NO: If The chromosome DNA of E. coli BL21 strain (Novagen, UK) was used as a template for PCR amplification of mazF with forward primer 5'-CAT ATG GTA AGC CGA TAC GTA CC-3' (SEQ ID NO: 70) and reverse primer 5'-AAG CTT CTA CCC AAT CAG TAC GTT AA-3’ (SEQ ID NO: 33) The PCR product, encoding mazF, and plasmid pET- 22b(+) were both digested with restriction enzymes TVcoI and Hindllf and then the fragments were ligated with T4 ligase (Fermentas) at 22°C overnight. The ligase mixture was transformed into E. coli XLlBlue cells and grown on LB agar plates with 100 pg/ml ampicillin overnight. The obtained colonies were verified with PCR for pelBmazF sequence and one of positive clones was inoculated and cultivated in LB medium with 100 pg/ml ampicillin for further isolation of plasmid pet22b-T7pelBmazF. The selected mutation to T7 promoter sequence (T448, TAATACGACTCTCTATAGGGAGA, SEQ ID NO: 26) was introduced by site- directed mutagenesis with forward primer 5'-agatcttaatacgactctctatagg-3' (SEQ ID NO: 18) and reverse standard primer, 5’-atgctagttattgctcag-3’ (SEQ ID NO: 27). The obtained PCR-product was ligated to pet22b plasmid by Bgl II - Hind III sites with T4 ligase (Fermentas). The ligase mixture was transformed to E. coli XllBlue cells and grown on LB agar plates +100 pg/ml ampicillin overnight. The positive clones were inoculated in LB+100 pg/ml ampicillin for plasmid pet22b-T448pelBMazF purification. [00370] Assembly of constructs for chromosome integration. The dsDNA T448pelBMazF- kanR cassette was synthesized by ligation of two PCR products by BamHl restriction site (PCR1 : T448pelBMazF from pet22b-T448pelBMazF with forward primer, containing 65 bp homology to flagA gene and 24 bp homology to plasmid pet22b-T448pelBMazF (5 - CTC CAA ATA CAC CAA AGC AAT GTA TAT GGA TCT GCT GGC TCT GCT TTA TCG GTT GAT GGC GAA ATC ACG ATG CGT CCG GCG TAG AGG ATC G-3 , SEQ ID NO: 46) and reverse primer with BamHl restriction site at the end (5 - GGA TCC ATC CGG ATA TAG TTC CTC CTT TCA GCA - 3, SEQ ID NO: 47); PCR2 - kanamycin resistant gene kanR - from pGBKT7 with forward primer with BamHl restriction site (5 - GGA TCC CGG GGA AAT GTG CGC GGA ACC-3 , SEQ ID NO: 48) and reverse primer containing 65 bp flank to flagA gene and 24bp homology to plasmid pGBKT7 (5 - TTCTGACGTAAAACAGTCGCTAATGGGGAAATAAATCCGTAAGCCAATAAAATG CCGAGGAAAGTACCCCAGAGTCCCGCTCAGAAGAACT-3 , SEQ ID NO: 49). Both PCR fragments were digested with BamHl and ligated with T4 ligase. Further amplification of ligase mixture with flanked primers resulted in T448pelBmazF-kanR product, which was purified by separation by gel electrophoresis in agarose gel followed by band excision and DNA release with spin-columns (Eurogene). The resultant product of 1765 bp was prepared for recombination. The scheme of assembly of T448pelBmazF-kanR construct for chromosome integration is shown in Figure 1.

[00371] Deletion of chromosomal module mazEF operon. The chromosomal module mazEF was deleted by its replacement with a sequence of streptomycin resistant gene (StmR) flanked with attL (5’- tga age etg ett ttt tat act aag ttg gca tta taa aaa age att get tat caa ttt gtt gca acg aac agg tea eta tea gtc aaa ata aaa tea tta ttt gat ttc - 3’, SEQ ID NO: 50) and attR (5 - ege tea agt tag tat aaa get gaa ega gaa acg taa aat gat ata aat ate aat ata tta aat tag att ttg cat aaa cag act aca taa tac tgt aaa aca caa cat atg cag tea eta tga ate aac tac tta gat ggt att agt gac etg taa cag a-3’, SEQ ID NO: 51) sequences. It was amplified from the plasmid pISA with primers containing 60 bp homology to the integration site on the chromosome, HR MazEF/attL Sp (5'- ATGATCCACAGTAGCGTAAAGCGTTGGGGAAATTCACCGGCGGTGCGGAT CCCGGCTACGTTAATGATCCTCTAGAGTCGAGATCTTGA-3', SEQ ID NO: 28) and HR MazEF/attp Sp (5¹-

CTACCCAATCAGTACGTTAATTTTGGCTTTAATGAGTTGTAATTCCTCTGGG GCAACTGTTCCTCAGGTCGACTAGACGCTCAA-3, SEQ ID NO: 29). The PCR product was separated by electrophoresis and gel purified. Colonies of E coli T7 Express Iq already containing pkD46 plasmid (GenBank: AY048746.1) were inoculated to 10 ml of fresh LB supplemented with 100 pg/ml of ampicillin and grown at 30°C overnight. Culture was diluted 100-fold in lOmL of LB medium and grown to ODeoo 0.2 at 30°C. L-arabinose was added to 0.2% final concentration to induce red expression, and the culture was grown to ODeoo = 0.6. The cell were harvested with centrifugation at 4°C, resuspended in 1 mL cold water, washed, pelleted, and resuspended in 500 pl of water. 10 pl of PCR product containing 500 ng of stmR targeted to MazEF region was added to 50pl of cell and the cells were electroporated. The culture was recovered in LB (1 mL) for 3 h, and then spread on LB agar supplemented with 50 pl streptomycin plates and grown overnight. Resultant recombinants were plated on fresh LB agar with 100 pg/ml ampicillin for negative selection of transformants with pkD46 plasmid. The obtained colonies were streaked on fresh LB agar plates with streptomycin.

[00372] Integration of T448pelBmazF-kanR into E.coli T7 Express Iq chromosome. Integration of the T448pelBMazF fused with the kanR kanamycin resistance gene for selection of recombinants into the E. coli T7 Express Iq AmazEF chromosome was performed using the Red recombination system of the bacteriophage lambda Red in two steps using conventional methodology by those skilled in the art. The genes for recombination proteins (lambda exonuclease, exo; beta protein, bet; gamma protein, gam) are contained in the pkD46 plasmid under an inducible arabinose promoter.

[00373] StmR removal was performed with integration (int) and cleavage (xis) proteins of phage X site-specific recombination from plnt/Xis plasmid under the inducible Plac promoter. plntXis was transformed to E.coli T7 Express Iq mazEF::stmR culture and transformants were plated to LB agar with ampicillin and IPTG and grown at 37°C overnight. Final selection of transformants was made by replating colonies on selective media:

- LB agar, 42°C, plnt/Xis is removed;

- LB agar+ stm for negative selection of StmR colonies;

- LB agar for colonies no longer containing StmR.

[00374] The obtained clones were modified as E. coli T7 Express Iq AmazEF.

[00375] Chromosomal integration of T448pelBmazF-kanR cassette. E. coli T7 Express AmazEF pkD46 culture was grown in LB medium at 30°C overnight. The culture was diluted 100-fold in 10 mL of LB medium and grown to ODeoo 0.2 at 30°C, then L-arabinose was added up to 0.2% final concentration to induce red expression, and the culture was grown to ODeoo 0.6. The cells were harvested with centrifugation at 4°C, resuspended in 10 mL of cold water and washed, pelleted, and resuspended in 500 pl of water. 10 pl of PCR product containing 500 ng of dsDNA T448pelBMazF-kanR targeted to flagA was added to 5 Opl of cell and the cells were electroporated. The culture was recovered in LB (ImL) for 3 hours, and then spread on LB agar supplemented with 50pl kanamycin plates and grown overnight. The resultant recombinants were plated to fresh LB with 100 pg/ml ampicillin for negative selection of transformants with pkD46 plasmid. Colonies, no longer containing pkD46, were streaked on fresh LB agar plates with 50 pg/ml of kanamycin. The resulting clones had an integrative insert on the flagA::T448pelBmazF-kanR chromosome, that was confirmed by sequencing. The obtained strain was named E. coli T7 Express A.

[00376] Assembly of a construct for MazF secretion with Tor A signal peptide. The construct in which mazF sequence was fused to twin-arginine signal peptide of TMAO reductase (TorA) was assembled for MazF export by the Tat pathway. The TorA Tat signal peptide (1-39 aa, MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA, SEQ ID NO: 2) with four residues AQAA (40-43 aa) (SEQ ID NO: 71) of the mature protein (1-43 aa, MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATAAQAA, SEQ ID NO: 25) was amplified from the E.coli BL21 chromosome with primers TorA-fwd (5 - aaacatatgaacaataacgatctctttcag-3’, SEQ ID NO: 30) and TorA-rev (5’- aaagcatgcgccgcttgcgccgcagt-3 ’ , SEQ ID NO: 31), and then digested with NdEl and BamHl enzymes. The mazF sequence was amplified from E. coli BL21 chromosome with primers 5’- aaagcatgcgtaagccgatacgtacc-3 ’ (SEQ ID NO: 32) and 5'-aagcttctacccaatcagtacgttaa-3' (SEQ ID NO: 33), and later digested with Sphl and Hindlll enzymes. The both fragments were cloned into NdEl -Hindlll sites of pet22b-T448pelBMazF plasmid with T4 ligase (Fermentas) and the mixture was transformed into E. coli XLlBlue cells to grow overnight on LB agar plates with 100 pg/ml ampicillin. The obtained colonies were PCR tested with TorA-fwd and TorA-rev primers to confirm the construction pet22b- T448TorAmazF. Nucleotide sequence(s) was confirmed by sequencing.

[00377] The plasmids pet22b-T448torAmazF and pGBKT7 were used for dsDNA T448torAmazF-kanR assembly of 1827 bp, as shown in Figure 2. The T448torAmazF-KanR cassette was integrated into E coli T7 Express \mazEF chromosome to produce the mutant strain named E. coli T7 Express B. The integration was confirmed by sequencing. The integration method with Red-lambda recombination proteins was used as described above. [00378] Assembly of a construct for secreted MazF expression under control of a te promoter. The tc_pelBmazF construct was assembled as follows. Three dsDNA fragments were linked by Gibson assembly (Figure 3). The nucleotide sequence of TetR gene (with primers 5 - TTAAGACCCACTTTCACATTTAAGTTG-3 , SEQ ID NO: 52; and 5’- ATCAATGATAGAGTGTCAACGAATTAATGATG-3 , SEQ ID NO: 53) and Tet operon (with primers 5 -GTTGACACTCTATCATTGATAGAGT-3 , SEQ ID NO: 54; and 5 - CTTAAAGTTAAACAAAATTATTTTCTCTATCACTGATAGGGAGTG-3 , SEQ ID NO:55) were consistently amplified from pASK-IBA4 plasmid (IBA Lifescience GmbH, Germany). The nucleotide sequence of pelBMAzF was amplified from pet22b-T448pelBmazF plasmid (5 -AATAATTTTGTTTAACTTTAAGAAGGAGATAT-3 , SEQ ID NO: 56; and 5 - TCAGTGGTGGTGGTGGTGGTGCTCGAGT-3 , SEQ ID NO: 57). The primers had 20 bp overlaps. Three PCR products were incubated 15 min at 50°C and ligase mixture was amplified with external primers with Encyclo polymerase for further ligation into pal2-T vector by T-A sticky ends (22°C, overnight). The ligase mixture was transformed into E. coll XLlBlue cells and one of transformants (verified with PCR, M13+/- primers 5 -GTT GTA AAA CGA CGG CCA GTG-3 ’, SEQ ID NO: 67; and 5 -AGC GGA TAA CAA TTT CAC AC A GGA- 3 , SEQ ID NO: 68) was inoculated and cultivated in LB medium with 100 pg/ml ampicillin for further isolation of the plasmid pal2T-tc_pelBmazF.

[00379] For integration into the E. coll. T7 Express Iq AmazEF chromosome, tc_pelBmazF construct was fused with the kanR kanamycin resistance gene (pGBKT7 plasmid). Selection of recombinants was carried out using the Red recombination system of the bacteriophage lambda Red (pkD46 plasmid) as described above. Sequence(s) of the resultant strain T7 Express Iq AmazEF flagA::tc_pelBmazF-kanR was confirmed by sequencing and named E. coli T7 Express C.

[00380] Construction of strain producers of recombinant human parathyroid hormone (hPTH) using MazF technology. An hPTH gene transcribed into ACA-less mRNAs was synthesized chemically. The precursor of hPTH (SEQ ID NO: 59) contains 115 amino acids (encoded by hPTH nucleotide sequence for E. coli, SEQ ID NO: 58) and the mature hPTH contains 84 amino acids (SEQ ID NO: 61); its nucleotide sequence contains 5 ACA sites. The gene encoding mature hPTH was synthesized from 10 single-strain oligonucleotides with 100- 110 nucleotides, each with overlapping ends (Table 3). The oligonucleotides were melted and annealed into double-stranded molecules. Codon composition of the hPTH nucleotide sequence (SEQ ID NO: 60) was optimized for E. coli expression and ACA sites in both the protein-encoding region and 5 ’-3’ non-translated regions in the pet32a vector were replaced while retaining the amino acid sequence (SEQ ID NO: 61).

[00381] The gene was assembled as follows: Every internal segment had overhanging 5’ and 3’ ends, complementary to the neighboring segment. For PCR, the double-stranded segments were generated from single-stranded oligonucleotides (pairs 1-2, 3-4, 5-6, 7-8, 9-10) and the single-strand gaps were annealed with Gibson Assembly mix (NEB, UK), which contained both the ligase and polymerase. Upon assembly, the gene was amplified by PCR with hPTH forward primer (SEQ ID NO: 44) and hPTH reverse primer (SEQ ID NO: 45) up to the quantity sufficient for ligation into pet32a (Novagen, UK) by Xbal and BamHI restriction sites. The ligase mix was transformed into 73 coll XLlBlue cells by CaCh transformation. Plasmid DNA was prepared from the resulted clones and verified by sequencing (Figure 4).

Table 3. Single strand oligonucleotides for assembly of the synthetic human PTH.

[00382] Expression of recombinant human parathyroid hormone (hPTH). Plasmid DNA pet32a-hPTH was transformed into the E. coli T7 Express A strain. The transformed strain grew on LB agar with kanamycin and ampicillin. The grown clones were verified with PCR with vector-specific primers (hPTH forward, hPTH reverse, Table 3). The selected positive clones were plated on fresh LB agar again with kanamycin and ampicillin.

[00383] Overnight cultures were refreshed by diluting 1 : 100 in 25 ml of LB medium (kanamycin and ampicillin added) up to ODeoo 0.05 and grown in the flasks on shaker at 37°C. Expression of MazF and hPTH was induced by 1 mM IPTG when the optic density reached ODeoo = 0.150. The flasks were allowed to grow for 7 hours after induction.

[00384] 1 -ml cell suspension samples for evaluation of soluble hPTH production were taken at 4 and 6 hours after induction. The cells were collected by centrifugation and supernatant was discarded. The pellet was dissolved in 3 ml of phosphate buffered saline (PBS) and sonicated on ice for 15 min (20 sec burst at 300 W, 15 sec cooling period). The lysate was centrifuged to pellet the cell debris. Soluble cellular proteins were analyzed by SDS-PAGE followed by Coomassie Blue staining.

Example 1. Effect of secreted MazF (PelBMazF) expression from an attenuated T7 promoter on microbial growth of a culture growing in a mineral medium.

[00385] A single colony from an E. coli T7 Express A culture freshly plated on solid Luria- Bertani (LB) medium was inoculated into a 250 ml Erlenmeyer flask containing 25 ml of synthetic M9 medium supplemented with 10 g/L glucose and 50 pg/ml kanamycin and grown overnight at 37°C on a shaker-incubator at 250 rpm. 0.25 ml of the obtained overnight culture was used to inoculate each of the 250 ml Erlenmeyer flasks containing 25 ml of M9 medium supplemented with 10 g/L glucose and 50 pg/ml kanamycin. The cultures were incubated at 37°C on a shaker at 250 rpm. Microbial growth was monitored by measuring optical density of the cultures at 600 nm every 60 min. PelBMazF synthesis was induced in E. coli T7 Express A cells by addition of a filter-sterilized Isopropyl [3-d-l -thiogalactopyranoside (IPTG) solution at 90 min of incubation. Cultures of E. coli T7 Express Iq and E. coli AT7 Exp del T448mazF- kanR prepared according to the same procedure were used as controls. Kanamycin and IPTG were not added to E. coli T7 Express Iq cultures. Both the experimental and control cultures were cultivated in triplicates. Dry cell weight was determined by filtration of liquid cultures through nitrocellulose membrane filters, pore size 0.2 m, followed by drying at 105°C to a constant weight.

[00386] Growth curves presented in Figure 5A show optical density measurements after additions of different concentrations of IPTG to E. coll T7 Express A. The obtained growth curves demonstrate that induction of PelBMazF expression by addition of 0.1 - 1.0 mM IPTG inhibited biomass accumulation in E. coll T7 Express A cultures in comparison with the E. coll T7 Express Iq control culture. However, a higher biomass accumulation in comparison with the control was recorded when 0.01 mM IPTG was added to an E. coll T7 Express A culture.

[00387] Exemplar growth curves presented in Figure 5B show means and standard deviations of optical density measurements in three biological replicates of both experimental and control E. coll cultures throughout their growth. The PelBMazF expression was induced in E. coll T7 Express A and E. coli AT7 Exp del T448mazF-kanR cultures by addition of IPTG in concentration of 0.01 mM. The cultures of E. coli T7 Express A and E. coli T7 Express Iq reached maximum of optical density after 8.5 hours of growth, however the maximal ODeoo of the strain E. coli T7 Express A was 11% higher than that of the strain E. coli T7 Express Iq, indicating that expression of secreted MazF protein in microbial cells under described conditions resulted in an increase in biomass yield by 11%. The cultures of E. coli AT7 Exp del T448mazF-kanR reached maximum of optical density after 10.5 hours of growth. The maximal ODeoo of these cultures was by 25% lower than that of the cultures of E. coli T7 Express A and by 17% lower than that of the cultures of E. coli AT7 Exp del T448mazF-kanR, indicating that the positive effect of PelBMazF expression was achieved due to establishing a MazF outflow from the cytoplasm. Otherwise, MazF accumulates in the cytoplasm and suppresses microbial growth. The results of the ODeoo measurements were confirmed by measurements of dry cell weight (DCW) in the samples of cultural liquids taken at the points of the maximal biomass accumulation (Table 4).

Table 4. Maximal biomass accumulation (DCW) in shake-flask cultures of E. coli T7 Express A producing PelBMazF presented as means of measurements in three biological replicates.

*E. coli T7 Express Iq and E. coli AT7 Exp del T448mazF-kanR were used as controls. [00388] Expression of the secreted fusion protein PelBMazF from promoters weaker than the T448 promoter (strength - 0.23 of T7), such as B93 (strength - 0.01 of T7) and B139 (strength

- 0.03 of T7) (see Table 1), did not result in any apparent modifications in the shape of the E. coll T7 Express A growth curve, /.< ., did not cause any apparent changes in the cellular metabolism. Therefore, promoters B93 and B139 were not used in further experiments with expression of secreted MazF. Expression of PelBMazF from promoters stronger than the T448 promoter, such as B 121 (strength - 0.30 of T7), H9 (strength - 0.35 of T7), and B 14 (strength

- 0.74 T7) (see Table 1) resulted in growth inhibition of the E. coli T7 Express A after IPTG additions to final concentrations of 0.01 - 1.0 mM. Therefore, promoters B121, H9, and B14 were not used in further experiments with expression of secreted MazF.

Example 2. Effect of secreted MazF (TorAMazF) expression from an attenuated T7 promoter on microbial growth of a culture growing in a mineral medium.

[00389] A single colony from an E. coli T7 Express B culture freshly plated on solid Luria- Bertani (LB) medium was inoculated into a 250 ml Erlenmeyer flask containing 25 ml of synthetic M9 medium supplemented with 10 g/L glucose and 50 pg/ml kanamycin and grown overnight at 37°C on a shaker-incubator at 250 rpm. 0.25 ml of the obtained overnight culture was used to inoculate each of the 250 ml Erlenmeyer flasks containing 25 ml of M9 medium supplemented with 10 g/L glucose and 50 pg/ml kanamycin. The cultures were incubated at 37°C on a shaker at 250 rpm. Microbial growth was monitored by measuring optical density of the cultures at 600 nm every 60 min. TorAMazF synthesis was induced in A. coli T7 Express B cells by addition of a filter-sterilized Isopropyl [3-d-l -thiogalactopyranoside (IPTG) solution at 90 min of incubation. Cultures of E. coli T7 Express Iq prepared according to the same procedure were used as controls. Kanamycin and IPTG were not added to E. coli T7 Express Iq cultures. Both the experimental and control cultures were cultivated in triplicates. Dry cell weight was determined by filtration of liquid cultures through nitrocellulose membrane filters, pore size 0.2 pm, followed by drying at 105°C to a constant weight.

[00390] Growth curves presented in Figure 6A show optical density measurements after additions of different concentrations of IPTG to E. coli T7 Express B. The obtained growth curves demonstrate that induction of PelBMazF expression by addition of 0.1 - 1.0 mM IPTG inhibited biomass accumulation in E. coli T7 Express B cultures in comparison with the E. coli T7 Express Iq control culture. However, a higher biomass accumulation in comparison with the control was recorded when 0.01 mM IPTG was added to an E. coli T7 Express B culture. [00391] Exemplar growth curves presented in Figure 6B show means of optical density measurements in three biological replicates of both experimental and control E. coli cultures throughout their growth. The TorAMazF expression was induced in E. coli T7 Express B cultures by addition of IPTG in concentration of 0.01 mM. The cultures of E. coli T7 Express B reached maximum of optical density after 9.5 hours of growth, however the maximal ODeoo of the strain E. coli T7 Express B was 16% higher than that of the strain E. coli T7 Express Iq, indicating that expression of secreted MazF protein in microbial cells under described conditions resulted in an increase in biomass yield by 16%. The results of the ODeoo measurements were confirmed by measurements of dry cell weight (DCW) in the samples of cultural liquids taken at the points of the maximal biomass accumulation (Table 5).

Table 5. Maximal biomass accumulation (DCW) in shake-flask cultures of E. coli T7 Express B producing PelBMazF presented as means of measurements in three biological replicates.

E. coli T7 Express Iq was used as control.

Example 3. Effect of secreted MazF (PelBMazF) expression from a Tc-inducible promoter on microbial growth of a culture growing in a mineral medium.

[00392] A single colony from an E. coli T7 Express C culture freshly plated on solid Luria- Bertani (LB) medium was inoculated into a 250 ml Erlenmeyer flask containing 25 ml of synthetic M9 medium supplemented with 10 g/L glucose and 50 pg/ml kanamycin and grown overnight at 37°C on a shaker-incubator at 250 rpm. 1.0 ml of the obtained overnight culture was used to inoculate each of the 250 ml Erlenmeyer flasks containing 50 ml of M9 medium supplemented with 10 g/L glucose and 50 pg/ml kanamycin. The cultures were incubated at 37°C on a shaker at 250 rpm. Microbial growth was monitored online by backscattering light detection by automated analysis system Cell Growth Quantifier (CGQ), (Aquilla Biolabs, Baesweiler, Germany). The online generated data was converted into ODeoo units according to the method provided by the manufacturer (aquila-biolabs.de/wp- content/uploads/2020/07/CGQ-User-Guide-rev-6_print.pdf). Dry cell weight was determined by filtration of liquid cultures through nitrocellulose membrane filters, pore size 0.2 pm, followed by drying at 105°C to a constant weight. PelBMazF synthesis was induced in E. coli T7 Express C cells by addition of a filter-sterilized solution of anhydrotetracycline hydrochloride (ATc) to a desired concentration at ODeoo = 1. The control culture of E. coli T7 Express Iq was prepared according to the same procedure but kanamycin and ATc were not added. Both the experimental and control cultures were cultivated in triplicates.

[00393] Exemplar growth curves presented in Figure 7 show means of optical density calculations based on on-line monitoring of microbial growth by a backscattering light detector in three biological replicates of both experimental E. coli T7 Express C and control E. coli T7 Express Iq cultures throughout their growth. The PelBMazF expression was induced in E. coli T7 Express C cultures by addition of ATc in concentrations of 500 - 2000 ng/ml when ODeoo of the cultures reached 1.0 (approximately 3 h of cultivation). The obtained growth curves demonstrate that induction of the PelBMazF expression in the E. coli T7 Express C cells by addition of 500 and 800 ng/ml ATc did not significantly affect biomass accumulation though these growth curves reached their plateaus 3 h later than the one of the control culture. However, substantially higher biomass accumulation was recorded (15% compared to control) when ATc concentration was increased up to 1000 ng/ml. In this case, the maximum cell density of the recombinant strain was reached 7 h later than in the control culture. Further increases in the ATc dose to 1500 and 2000 ng/ml resulted in the partial growth inhibition. The results of the on-line measurements of cell density were confirmed by off-line measurements of dry cell weight (DCW) in the samples of cultural liquids taken at the points of the maximal biomass accumulation (Table 6).

Table 6. Maximal biomass accumulation (DCW) in shake-flask cultures of E. coli T7 Express C producing PelBMazF presented as means of measurements in three biological replicates.

E. coli T7 Express Iq was used as a control.

Example 4. Effect of secreted MazF (PelBMazF) expression from a Tc-inducible promoter on microbial growth of a culture growing in a complex medium.

[00394] A single colony from an E. coli T7 Express C culture freshly plated on solid Luria- Bertani (LB) medium was inoculated into a 250 ml Erlenmeyer flask containing 25 ml of Luria- Bertani (LB) medium and 50 pg/ml kanamycin and grown overnight at 37°C on a shakerincubator at 250 rpm. 1.0 ml of the obtained overnight culture was used to inoculate each of the 250 ml Erlenmeyer flasks containing 50 ml of Luria-Bertani (LB) medium and 50 pg/ml kanamycin. The cultures were incubated at 37°C on a shaker at 250 rpm. Microbial growth was monitored online by backscattering light detection by automated analysis system Cell Growth Quantifier (CGQ), (Aquilla Biolabs, Baesweiler, Germany). The online generated data was converted into ODeoo units according to the method provided by the manufacturer (aquila- biolabs.de/wp-content/uploads/2020/07/CGQ-User-Guide-rev-6_print.pdf). Dry cell weight was determined by filtration of liquid cultures through nitrocellulose membrane filters, pore size 0.2 pm, followed by drying at 105°C to a constant weight. PelBMazF synthesis was induced in E. coll T7 Express C cells by addition of a filter-sterilized solution of anhydrotetracycline hydrochloride (ATc) to a desired concentration at ODeoo = 1. The control culture of E. coll T7 Express Iq was prepared according to the same procedure but kanamycin and ATc were not added. Both the experimental and control cultures were cultivated in triplicates.

[00395] Exemplar growth curves presented in Figure 8 show means of optical density calculations based on on-line monitoring of microbial growth by a backscattering light detector in three biological replicates of both experimental E. coli T7 Express C and control E. coli T7 Express Iq cultures throughout their growth. The PelBMazF expression was induced in E. coli T7 Express C cultures by addition of ATc in concentrations of 500 ng/ml (Figure 8A) and 1000 ng/ml (Figure 8B) when ODeoo of the cultures reached 1.0 (approximately 3 h of cultivation). The obtained growth curves demonstrate that induction of the PelBMazF expression in the E. coli T7 Express C cells by addition of 500 and 1000 ng/ml ATc resulted in 28% and 26% biomass increase, correspondingly. The recombinant culture grew 8 h longer compare with the control strain in the both cases. The results of the on-line measurements of cell density were confirmed by off-line measurements of dry cell weight (DCW) in the samples of cultural liquids taken at the points of the maximal biomass accumulation (Table 7).

Table 7. Maximal biomass accumulation (DCW) in shake-flask cultures of E. coli T7 Express C producing PelBMazF presented as means of measurements in three biological replicates.

E. coli T7 Express Iq was used as a control. Example 5. Effect of secreted MazF (PelBMazF) expression on production of an exemplary target recombinant protein.

[00396] Cells of E. coli T7 Express Iq and E. coli T7 Express A were transformed with a high- copy-number plasmid pet32a containing an Adenine-Cytosine-Adenine (ACA)-less nucleotide sequence, /.< ., a nucleotide sequence lacking an ACA sequence(s), encoding the human parathyroid hormone (hPTH) protein and the ampicillin-resistance gene.

[00397] Single colonies of E. coli T7 Express Iq pet32a-hPTH and E. coli T7 Express A pet32a-hPTH from cultures freshly plated on solid LB media supplemented with 100 mg/L ampicillin (the medium for growth of E. coli T7 Express A pet32a-hPTH was also supplemented with 50 pg/ml kanamycin) were inoculated into 250 ml Erlenmeyer flasks containing 25 ml of LB medium supplemented with 50 pg/ml kanamycin (for E. coli T7 Express A pet32a-hPTH) and 100 mg/L ampicillin. The cultures were grown overnight at 37°C in a shaker-incubator at 250 rpm.

[00398] 0.25 ml of the obtained overnight cultures were used to inoculate 250 ml Erlenmeyer flasks containing 25 ml of LB medium supplemented with 50 pg/ml kanamycin (for A. coli T7 Express A pet32a-hPTH) and 100 mg/L ampicillin. The cultures were incubated at 37°C in a shaker-incubator at 250 rpm. When the optic density reached ODeoo = 0.150 (~ 90 min of incubation), a filter-sterilized solution of IPTG was added to the cultures to a final concentration of 1.0 mM to induce synthesis of hPTH. These IPTG additions also induced synthesis of secreted MazF (PelBMazF) in the culture of E. coli T7 Express A pet32a-hPTH.

[00399] 1 -ml cell suspension samples for evaluation of soluble hPTH production were taken at 4 and 6 hours after induction. The cells were collected by centrifugation and supernatant was discarded. The pellet was dissolved in 3 ml of phosphate buffered saline (PBS) and sonicated on ice for 15 min (20 sec burst at 300 W, 15 sec cooling period). The lysate was centrifuged to pellet the cell debris. Soluble cellular proteins were analyzed by SDS-PAGE followed by Coomassie Blue staining.

[00400] Growth curves presented in Figure 9 show ODeoo measurements throughout the cultivation of A. coli T7 Express A pet32a-hPTH and A. coli T7 Express Iq pet32a-hPTH. The obtained growth curves demonstrate that the simultaneous induction of PelBMazF and hPTH expressions by addition of 1.0 mM IPTG resulted in a significantly inhibited biomass accumulation of E. coli T7 Express A pet32a-hPTH in comparison with the E. coli T7 Express Iq pet32a-hPTH control culture. Biomass accumulation of E. coli T7 Express A pet32a-hPTH was 52% of that of E. coli T7 Express Iq pet32a-hPTH. [00401] The hPTH expression ratio between the two strains was determined at 4 and 6 hours after induction. Densitometric quantification of the protein loads stained with Coomassie Brilliant Blue demonstrated that E. coll T7 Express A pet32a-hPTH produces 7.1 times more of soluble hPTH than E. coli T7 Express Iq pet32a-hPTH after 4 h of induction and 9.8 times more after 6 h of induction.

Example 6. MazEF toxin-antitoxin (TA) module in E. coli cells as a metabolic switch.

[00402] This example provides support for the natural function of the MazEF TA module in E. coli cells as a metabolic switch to transition from a “feast” phenotype to a “hunger” one before cells enter into the stationary phase.

[00403] Bacterial growth. E. coli MG1655 (CGSC 6300; E. coli Genetic Stock Center, Yale Science Building Rm 335, Dept, of Molecular, Cellular, and Developmental Biology, 266 Whitney Avenue, Yale University, New Haven, CT 06520-8103 ) was grown on LB medium (5 g yeast extract, 10 g peptone tryptone, 10 g NaCl; Miller, 1972) in batch culture for 8 hours. Cultivation was carried out at 37°C in a laboratory-scale Bioflo 2000 (New Brunswick Scientific) fermentor with a 2 L working volume. The pH was maintained at 6.95 automatically by titration with 5% H2SO4 or 5% NaOH. Dissolved oxygen (DO) in the culture was maintained at 40% saturation automatically by varying speed of impeller rotation.

[00404] Samples for ODeoo analysis were taken every 30 min. The growth curve and specific growth rate (p) calculated for each 30-min interval between the samples are shown in Figure 10A. The maximum growth rate p_max = 1.68 h^-1 was observed between 1.5 and 2 hours of fermentation. This value is almost identical to that reported by others for E. coli MG1655 growing in LB broth (Polen et al., 2003). The growth rate decreased slightly but stabilized at a high level of 1.33-1.47 h^-1 for the next 1.5 h. Between 3.5 and 4 hours of fermentation, p dropped almost two-fold to 0.82 h^-1 and slowly diminished to 0.53 h^-1 by 5 h of fermentation. During the next 30 min, p dropped fivefold to 0.11 h^-1, and by 7 h of fermentation, growth stopped completely.

E. coli DNA microarrays. E. coli genomic DNA from MG1655 was used as template for generating the microarray probe library. The ERGO database (Integrated Genomics) identifies 4,485 open reading frames (ORFs) within the E. coli genome; primers were designed using proprietary software such that the most unique 300-500 bp region of 4,442 of these ORFs was amplified with two consecutive rounds of polymerase chain reaction (PCR). PCR products were purified with an Array IT brand PCR Purification Kit (Telechem International) in 384- well format, dried, and resuspended in 15 pl of spotting buffer (45 mM sodium citrate, 0.45 M sodium chloride pH 7, 1.5 M betaine; Diehl et al. 2001) and printed in triplicate onto aminoalkylsilane coated slides (Sigma) with a GeneMachines OmniGrid arrayer (Genomic Solutions) using Telechem SMP3 split pins. The DNA was crosslinked to the slides with UV light. Residual salt and unbound DNA were removed by rinsing the slides with 0.5% SDS and water. [00405] Sample collection, target preparation, hybridization, and data analysis. Starting at 1.5 hours after inoculation, 10-25 ml samples of cell culture were withdrawn at 30-min intervals and mixed with 1 : 10 volume of ice-cold ethanol/phenol stop solution (5% water-saturated phenol, pH < 7.0 in absolute ethanol). Cells were harvested from the stop solution by centrifugation (10 min, 4,500*g, 4°C), flash-frozen in liquid nitrogen, and stored at -80°C. RNA was isolated with the RiboPure protocol (Ambion).

[00406] To obtain a reference RNA pool that contained all mRNA that was expressed at any time point of the fermentation, a mixture of equal portions of mRNA (26 pg) isolated from each sample was used as a reference for measuring differential expression. This strategy has been validated by microarray studies and provides a robust control for assessing relative changes in expression (Kerr, 2003). To obtain fluorescent target, 20 pg RNA was reverse transcribed with 500 U SuperScript II (Invitrogen) using random hexamer primers and a deoxyribonucleotide triphosphate mixture consisting of 0.5 mM dATP, dCTP, and dGTP each, 0.3 mM dTTP, and 0.2 mM amino-allyl dUTP (Sigma; described in detail at cmgm.stanford.edu/pbrown/protocols/ and by Khodursky et al., 2003). RNA was removed by alkaline hydrolysis (0.3 M NaOH, 15 min, 65°C).

[00407] After neutralization, cDNA was purified with QIAquick PCR Purification Kit (QIAGEN) and conjugated with the amino-reactive forms of Cy3 and Cy5 (Cy3 MonoReactive Dye Pack and Cy5 Mono-Reactive Dye Pack, Amersham Biosciences) as described in Cox and Singer, 2004. The reference sample was labeled with Cy3; cDNA from individual time points was labeled with Cy5. Unincorporated dye was removed by ultrafiltration on Microcon YM30 (Millipore). Two-microgram labeled reference and experimental cDNA was resuspended in 100 pl Sigma Array Hyb Low Temp Hybridization solution (Sigma), supplemented with 1 pg each of salmon sperm DNA and yeast tRNA. Hybridizations were performed in a GeneTAC Hyb Station (Genomic Solutions) overnight at 42°C. Prehybridization and stringency washes were conducted as described in Hedge et al., 2000.

[00408] Arrays were scanned on an Axon Instruments GenePix 4000B Array Scanner (Molecular Devices) at 635 and 532 nm. Raw images were analyzed using the GenePix Pro 3.0 software (Axon Instruments, Molecular Devices). Background subtracted data were compiled and organized within Microsoft Access. Gene-specific median of ratio data was extracted from the database and normalized to the highest expression level (arbitrarily assigned 100%). The plots related to mazF and mazE relative expression show means and standard deviations of three replicate measurements along the time course (Figure 10B).

[00409] The relative expression profile of mazF encoding the MazF toxin protein exhibits a single peak at 5.5 hours, which coincides with the 5 -fold drop of the culture growth rate upon entry into the “hunger” state. This coincidence of the peak in mazF expression with the entry into the “hunger” state distinguishes mazF expression curve from all the other 4,441 E. coll MG1655 gene expression curves obtained in this experiment. The peak in mazF relative expression is flanked by two peaks in relative expression of the mazE gene encoding the MazE antitoxin protein, which is known to neutralize the toxic effect exerted by MazF.

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[00410] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[00411]All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.

LIST OF SEQUENCES

SEQ ID NO: 1 PelB signal peptide

MKYLLPTAAAGLLLLAAQPAMA

SEQ ID NO: 2 TorA signal peptide 1-39 aa

MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA

SEQ ID NO: 3 H9 promoter

TAATACGACTCACTAATACTGAA

SEQ ID NO: 4 G6 promoter

TAATACGACTCACTATTTCGGAA

SEQ ID NO: 5 T448 promoter; forward primer, site-directed mutagenesis T7 promoter sequence

TAATACGACTCTCTATAGG

SEQ ID NO: 6 B14 promoter

TAATACGACTCACTATAGGAGAA

SEQ ID NO: 7 B121 promoter

TAATACCACTCACTATAGGGAGA

SEQ ID NO: 8 B282 promoter

TAATACAACTCACTATAGGGAGA

SEQ ID NO: 9 B233 promoter

TAATACGTCTCACTATAGGGGAA

SEQ ID NO: 10 T7 consensus

TAATACGACTCACTATAGGGGAA

SEQ ID NO: 11 B93

TAATACGACTCAATATAGGGAGA

SEQ ID NO: 12 B139

TAATACGACTCCCTATAGGGAGA

SEP ID NO: 13 T7 + Bgl II promoter

AGATCTTAATACGACTCACTATAGGGGAA

SEP ID NO: 14 H9 + Bgl II promoter

AGATCTTAATACGACTCACTAATACTGAA

SEP ID NO: 15 G6 + Bgl II promoter

AGATCTTAATACGACTCACTATTTCGGAA

SEP ID NO: 16 bgl + B93 promoter

AGATCTTAATACGACT

AATATAGG SEO ID NO: 17 bgl II + B139 promoter

AGATCTTAATACGACTCCCTATAGG

SEP ID NO: 18 bgl II + T448 promoter

AGATCTTAATACGACTCTCTATAGG

SEP ID NO: 19 bgl II + B57 promoter

AGATCTGAACTTAATACGACTCACTATAGG

SEQ ID NO: 20 _ bgl II + B14 promoter

AGATCTTAATACGACTCACTATAGGAGAA

SEQ ID NO: 21 bgl II + B121 promoter

AGATCTTAATACCACTCACTATAGGGAGA

SEQ ID NO: 22 bgl II + B282 promoter

AGATCTTAATACAACTCACTATAGGGAGA

SEQ ID NO: 23 bgl II + B233 promoter

AGATCTTAATACGTCTCACTATAGGGGAA

SEP ID NO: 24 T7 reverse

TAATACGACTCACTATAGG

SEQ ID NO: 25 TorA signal peptide 1-43 aa, mature

MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATAAQAA

SEQ ID NO: 26 _ T448 promoter

TAATACGACTCTCTATAGGGAGA

SEQ ID NO: 27 reverse primer, site-directed mutagenesis T7 promoter sequence

ATGCTAGTTATTGCTCAG

SEP ID NO: 28 HR MazEF/attL Sp

ATGATCCACAGTAGCGTAAAGCGTTGGGGAAATTCACCGGCGGTGCGGATCCCG

GCTACGTTAATGATCCTCTAGAGTCGAGATCTTGA

SEP ID NO: 29 HR MazEF/attp Sp

CTACCCAATCAGTACGTTAATTTTGGCTTTAATGAGTTGTAATTCCTCTGGGGCAA

CTGTTCCTCAGGTCGACTAGACGCTCAA

SEQ ID NO: 30 _ TorA-fwd primer

AAACATATGAACAATAACGATCTCTTTCAG

SEQ ID NO: 31 _ TorA-rev primer

AAAGCATGCGCCGCTTGCGCCGCAG

SEQ ID NO: 32 _ MazF primer, forward

AAAGCATGCGTAAGCCGATACGTACC SEQ ID NO: 33 _ MazF primer, reverse

AAGCTTCTACCCAATCAGTACGTTAA

SEQ ID NO: 34 hPTH 1

CATCGCCGGCTGGGCAGCGAGGAGCAGCAGACCAGCAGCAGCGGTCGGCAGCA

GGTATTTCATATCTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGA

SEQ ID NO: 35 hPTH 2

TCGCTGCCCAGCCGGCGATGGCCATGAGCGATAAAATTATTCACCTGACTGACG

ATAGTTTTGATACGGATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATT

SEQ ID NO: 36 hPTH 3

ACGGTCAGTTTGCCCTGATATTCGTCAGCGATTTCATCCAGAATCGGGGCGATCA

TTTTGCACGGACCGCACCACTCTGCCCAGAAATCGACGAGGATCGCCCCG

SEP ID NO: 37 hPTH 4

TATCAGGGCAAACTGACCGTTGCAAAACTGAATATCGATCAAAACCCTGGCACT

GCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAAC

SEP ID NO: 38 hPTH 5

AACCAGAACCGGCCAGGTTAGCGTCGAGGAACTCTTTCAACTGACCTTTAGACA

GTGCACCCACTTTGGTTGCCGCCACTTCACCGTTTTTGAACAGCAGCAGAG

SEP ID NO: 39 hPTH 6

TTGGTCTTGGCCGGTCCAATTTCGCTGACGCTCTTATCGTCGTCGTCGGTACCCAG

ATCTGGGCTATCCATATGCTGGCGTTCGTTAAATCGTCGTCGCCAAAG

SEP ID NO: 40 hPTH 7

CAGGTTATGCATCAGCTGAATTTCGCTGACGCTCTTATCGTCGTCGTCGGTACCC

AGATCTGGGCTATCCATATGCTGGCGTTCGAATTTAGCAGCAGCGGTTTC

SEP ID NO: 41 hPTH 8

TTCAGCTGATGCATAACCTGGGTAAGCATCTGAATAGCATGGAACGCGTTGAAT

GGCTGCGTAAGAAACTGCAGGATGTGCATAACTTTGTGGCGCTGGGCGCAC

SEP ID NO: 42 hPTH 9

CCCAGGCTTTTTTCATGGCTTTCCACCAGCACGTTATCTTCTTTTTTACGCGGACG

CTGGCTGCCCGCATCGCGCGGCGCCAGCGGTGCGCCCAGCGCCACAAAG

SEP ID NO: 43 hPTH 10

AGCCATGAAAAAAGCCTGGGCGAAGCGGATAAAGCCGATGTTAACGTTCTGACC

AAAGCGAAATCGCAGTAAGGATCC

SEP ID NO: 44 hPTH forward

TCTAGAAATAATTTTGTTTAACTTTAAGAAG

SEP ID NO: 45 hPTH reverse

GGATCCTTACTGCGATTTCG SEP ID NO: 46 PCR1 T448pelBMazF from pet22b-PCRl T448pelBMazF, forward primer

CTCCAAATACACCAAAGCAATGTATATGGATCTGCTGGCTCTGCTTTATCGGTTG ATGGCGAAATCACGATGCGTCCGGCGTAGAGGATCG

SEQ ID NO: 47 _ T448pelBMazF from pet22b-T448pelBMazF, reverse primer (with

BamHl restriction site at the end)

GGATCCATCCGGATATAGTTCCTCCTTTCAGCA

SEQ ID NO: 48 _ PCR2 - kanamycin resistant gene kanR - from pGBKT7, forward primer (with BamHl restriction site )

GGATCCCGGGGAAATGTGCGCGGAACC

SEQ ID NO: 49 PCR2 - kanamycin resistant gene kanR - from pGBKT7, reverse primer (containing 65 bp flank to flagA gene and 24bp homology to plasmid pGBKT7)

TTCTGACGTAAAACAGTCGCTAATGGGGAAATAAATCCGTAAGCCAATAAAATG CCGAGGAAAGTACCCCAGAGTCCCGCTCAGAAGAACT

SEQ ID NO: 50 streptomycin resistant gene (StmR) flanked with attL

TGAAGCCTGCTTTTTTATACTAAGTTGGCATTATAAAAAAGCATTGCTTATCAATT TGTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATTATTTGATTTC

SEQ ID NO: 51 _ streptomycin resistant gene (StmR) flanked with attR

CGCTCAAGTTAGTATAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATC AATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACAC

AACATATGCAGTCACTATGAATCAACTACTTAGATGGTATTAGTGACCTGTAACA GA

SEQ ID NO: 52 nucleotide sequence of TetR gene, primer

TTAAGACCCACTTTCACATTTAAGTTG

SEQ ID NO: 53 nucleotide sequence of TetR gene, primer

ATCAATGATAGAGTGTCAACGAATTAATGATG

SEQ ID NO: 54 _ Tet operon, primer

GTTGACACTCTATCATTGATAGAGT

SEQ ID NO: 55 _ Tet operon, primer

CTTAAAGTTAAACAAAATTATTTTCTCTATCACTGATAGGGAGTG

SEQ ID NO: 56 Nucleotide sequence pelBMAzF amplified from pet22b-

T448pelBmazF plasmid, primer

AATAATTTTGTTTAACTTTAAGAAGGAGATAT

SEQ ID NO: 57 Nucleotide sequence pelBMAzF amplified from pet22b-

T448pelBmazF plasmid, primer

TCAGTGGTGGTGGTGGTGGTGCTCGAGT

SEQ ID NO: 58 hPTH nucleotide sequence (for E coli) 345bp ATGATACCTGCAAAAGACATGGCTAAAGTTATGATTGTCATGTTGGCAATTTGTT

TTCTTACAAAATCGGATGGGAAATCTGTTAAGAAGAGATCTGTGAGTGAAATAC

AGCTTATGCATAACCTGGGAAAACATCTGAACTCGATGGAGAGAGTAGAATGGC

TGCGTAAGAAGCTGCAGGATGTGCACAATTTTGTTGCCCTTGGAGCTCCTCTAGC

TCCCAGAGATGCTGGTTCCCAGAGGCCCCGAAAAAAGGAAGACAATGTCTTGGT

TGAGAGCCATGAAAAAAGTCTTGGAGAGGCAGACAAAGCTGATGTGAATGTATT AACTAAAGCTAAATCCCAG

SEP ID NO: 59 hPTH Translation (115 aa)

MIPAKDMAKVMIVMLAICFLTKSDGKSVKKRSVSEIQLMHNLGKHLNSMERVEWL

RKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLT KAKSQ

SEQ ID NO: 60 Optimized hPTH nucleotide sequence (for E coli) 1-84 aa (252 bp)

AGCGTCAGCGAAATTCAGCTGATGCATAACCTGGGTAAGCATCTGAATAGCATG

GAACGCGTTGAATGGCTGCGTAAGAAACTGCAGGATGTGCATAACTTTGTGGCG

CTGGGCGCACCGCTGGCGCCGCGCGATGCGGGCAGCCAGCGTCCGCGTAAAAAA

GAAGATAACGTGCTGGTGGAAAGCCATGAAAAAAGCCTGGGCGAAGCGGATAA

AGCCGATGTTAACGTTCTGACCAAAGCGAAATCGCAG

SEQ ID NO: 61 _ Optimized hPTH Translation (84 aa):

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKE

DNVLVESHEI<SLGEADI<ADVNVLTI<AI<SQ

SEQ ID NO: 62 amino acid sequence of Maz F

MVSRYVPDMGDLIWVDFDPTKGSEQAGHRPAVVLSPFMYNNKTGMCLCVPCTTQS

KGYPFEVVLSGQERDGVALADQVKSIAWRARGATKKGTVAPEELQLIKAKINVLIG

SEQ ID NO: 63 _ MazF Escherichia coli str. K-12 substr. MG1655

ATGGTAAGCCGATACGTACCCGATATGGGCGATCTGATTTGGGTTGATTTTGACC

CGACAAAAGGTAGCGAGCAAGCTGGACATCGTCCAGCTGTTGTCCTGAGTCCTTT

CATGTACAACAACAAAACAGGTATGTGTCTGTGTGTTCCTTGTACAACGCAATCA

AAAGGATATCCGTTCGAAGTTGTTTTATCCGGTCAGGAACGTGATGGCGTAGCGT

TAGCTGATCAGGTAAAAAGTATCGCCTGGCGGGCAAGAGGAGCAACGAAGAAA

GGAACAGTTGCCCCAGAGGAATTACAACTCATTAAAGCCAAAATTAACGTACTG ATTGGGTAG

SEQ ID NO: 64 _ MazF Escherichia coli O157:H7 str. Sakai

ATGGTAAGCCGATACGTACCCGATATGGGCGATCTGATTTGGGTTGATTTTGACC

CGACAAAAGGTAGCGAGCAAGCCGGACATCGTCCGGCTGTTGTCCTGAGTCCGT

TCATGTACAACAACAAAACAGGTATGTGTCTGTGTGTTCCTTGTACAACGCAATC

AAAAGGATATCCGTTCGAAGTTGTTTTATCCGGTCAGGAACGTGATGGCGTAGCG

TTAGCTGATCAGGTAAAAAGTATCGCCTGGCGGGCAAGAGGAGCAACGAAGAA

AGGAACGGTTGCCCCAGAGGAATTACAACTCATTAAAGCCAAAATTAACGTACT GATTGGGTAG

SEQ ID NO: 65 amino acid sequence of Maz E

MIHSSVI<RWGNSPAVRIPATLMQALNLNIDDEVI<IDLVDGI<LIIEPVRI<EPVFTLAEL

VNDITPENLHENIDWGEPKDKEVW SEQ ID NO: 66 _ MazE Escherichia coli str. K-12 substr; MazE Escherichia coli

O157:H7 str. Sakai

ATGATCCACAGTAGCGTAAAGCGTTGGGGAAATTCACCGGCGGTGCGGATCCCG

GCTACGTTAATGCAGGCGCTCAATCTGAATATTGATGATGAAGTGAAGATTGACC

TGGTGGATGGCAAATTAATTATTGAGCCAGTGCGTAAAGAGCCCGTATTTACGCT

TGCTGAACTGGTCAACGACATCACGCCGGAAAACCTCCACGAGAATATCGACTG

GGGAGAGCCGAAAGATAAGGAAGTCTGGTAA

SEQ ID NO: 67 M13+/- primer forward

GTTGTAAAACGACGGCCAGT

SEQ ID NO: 68 M13+/- primer reverse

AGCGGATAACAATTTCACACAGGA

SEQ ID NO: 69 Tc inducible promoter

GTTGACACTCTATCATTGATAGAGTTATTTTACCACTC

SEQ ID NO: 70 MazF primer, forward

CATATGGTAAGCCGATACGTACC

SEQ ID NO: 71 TorA Tat signal peptide AQAA (40-43 aa)

AQAA

SEQ ID NO: 72 exemplar codons comprising ACA sequence

XXA-CAX

SEQ ID NO: 73 exemplar codons comprising ACA sequence

XAC-AXX

Claims

CLAIMS A genetically modified cell, said cell comprising a first polynucleotide encoding a fusion polypeptide comprising a global metabolic regulator, or a component or functional fragment or derivative thereof, operably linked to a signal peptide which enables secretion of the fusion polypeptide to the space outside of the cytoplasmic membrane of said cell. The genetically modified cell of claim 1, wherein the expression of the fusion polypeptide in said cell does not fully inhibit growth of said cell. The genetically modified cell of claim 1 or claim 2, wherein the growth rate of said cell during the expression of the fusion polypeptide is higher than 0. The genetically modified cell of any one of claims 1-3, wherein the global metabolic regulator operably linked to the signal peptide is a toxin component of a toxin and antitoxin (TA) module, or a functional fragment or derivative thereof. The genetically modified cell of claim 4, wherein the fusion polypeptide comprises the same toxin component as the toxin component of an endogenous TA module of said cell, and the activity of said endogenous TA module of said cell is eliminated. The genetically modified cell of any one of claims 1-5, further comprising one or more additional polynucleotides, said additional polynucleotide(s) encoding at least one polypeptide which is a biological substance or which participates in the production of the biological substance. The genetically modified cell of claim 6, wherein the first polynucleotide and/or the one or more additional polynucleotide(s) is modified to replace one or more nucleotide sequences in a corresponding mRNA(s) that are recognizable by said toxin component, or the functional fragment or derivative thereof, so that such mRNA(s) becomes resistant to the destruction by said toxin component. The genetically modified cell of any one of claims 1-7, wherein the cell is a microbial cell. The genetically modified cell of claim 8, wherein the microbial cell is a prokaryotic cell. The genetically modified cell of claim 9, wherein the prokaryotic cell is a bacterial cell. The genetically modified cell of claim 9 or claim 10, wherein the prokaryotic cell is from a genus selected from Nocardia, Acetobacter, Bacillus, Clostridium, Corynebacterium, Erwinia, Escherichia, Gluconacetobacter, Gluconobacter, Klebsiella, Lactobacillus, Lactococcus, Mannheimia, Propionibacterium, Pseudomonas, Ralstonia, Saccharopolyspora, Salmonella, Serratia, Streptomyces, Xanthomonas, and Zymomonas. The genetically modified cell of claim 11, wherein the prokaryotic cell is from a species selected from Acetobacter aceti, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus subtilis, Clostridium acetobutylicum, Clostridium butyricum, Clostridium thermoaceticum, Clostridium tyrobutyricum, Corynebacterium glutamicum, Erwinia citreus, Erwinia herbicola, Gluconacetobacter hansenii, Gluconobacter oxydans, Klebsiella oxytoca, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Mannheimia succinicip-roducens, Nocardia lactamdurans, Propionibacterium shermanii, Pseudomonas denitrificans, Ralstonia eutropha, Saccharopolyspora erythrea, Saccharopolyspora spinosa, Serratia marcescens, Streptomyces clavuligerus, Streptomyces griseus, Streptomyces lividans, Streptomyces roseosporus, Xanthomonas campestris, Zymomonas mobilis, Escherichia coli, Lactococcus lactis, Bacillus cereus, Salmonella typhimurium, and Pseudomonas fluorescens. The genetically modified cell of claim 12, wherein the prokaryotic cell is from a species selected from Escherichia coli, Lactococcus lactis, Bacillus subtilis, Bacillus cereus, Salmonella typhimurium, and Pseudomonas fluorescens. The genetically modified cell of claim 13, wherein the prokaryotic cell is from the species Escherichia coli. The genetically modified cell of any one of claims 8-14, wherein the signal peptide enables secretion of the fusion polypeptide to the periplasm of the microbial cell. The genetically modified cell of any one of claims 8-15, wherein the signal peptide enables secretion of the fusion polypeptide to the extracellular space of the microbial cell. The genetically modified cell of any one of claims 1-7, wherein the cell is a eukaryotic cell. The genetically modified cell of claim 17, wherein the eukaryotic cell is a fungal cell. The genetically modified cell of claim 17 or claim 18, wherein the eukaryotic cell is from a genus selected from Chrysosporium, Eremothecium (Ashbya), Rhizopus, Acremonium (Cephalosporium) , Aspergillus, Arxula, Blakeslea, Candida, Fusarium, Ganoderma, Hansenula, Kluyveromyces, Mortierella, Mucor, Pachisolen, Penicillium, Phaffia, Pichia, Saccharomyces, Schizosaccharomyces, Trichoderma, Umbelopsis, Yarrowia, and Zygosaccharomyces . The genetically modified cell of claim 19, wherein the eukaryotic cell is from a species selected from Acremonium chrysogenum, Arxula adeninivorans, Aspergillus awamori, Aspergillus chrysogenum, Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, Candida boidinii, Candida sorenensis, Blakeslea trispora, Chrysosporium lucknowense, Eremothecium (Ashbya) gossypii, Fusarium venenatum, Ganoderma lucidum, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Mortierella alpine, Mucor miehei, Pachysolen tannophilus, Penicillium brevicompactum, Penicillium chrysogenum, Phaffia rhodozyma, Pichia methanolica, Pichia stipitis, Rhizopus oryzae, Trichoderma reesei, Umbelopsis isabellina, Yarrowia lipolytica, Zygosaccharomyces bailii, Pichia pastor is, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. The genetically modified cell of claim 20, wherein the eukaryotic cell is from a species selected from Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. The genetically modified cell of any one of claims 1-21, wherein the signal peptide is a Sec-dependent signal peptide or a twin-arginine translocation (TAT)-dependent signal peptide. The genetically modified cell of claim 22, wherein the signal peptide is a Sec-dependent signal peptide selected from LamB, LTB, MalE, OmpA, PelB, PhoA, SpA, PhoE, OmpT, OmpF, and functional fragments or derivatives thereof. The genetically modified cell of claim 23, wherein the signal peptide is a PelB signal peptide or a functional fragment or derivative thereof. The genetically modified cell of claim 24, wherein the PelB signal peptide, or the functional fragment or derivative thereof, comprises the amino acid sequence MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 1). The genetically modified cell of claim 22, wherein the signal peptide is a TAT-dependent signal peptide selected from TorA, Tap, and functional fragments or derivatives thereof. The genetically modified cell of claim 26, wherein the signal peptide is a TorA signal peptide or a functional fragment or derivative thereof. The genetically modified cell of claim 27, wherein the TorA signal peptide, or the functional fragment or derivative thereof, comprises the amino acid sequence MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 2). The genetically modified cell of any one of claims 1-28, wherein expression of the fusion polypeptide is under control of a promotor. The genetically modified cell of claim 29, wherein the promoter is a constitutive promoter. The genetically modified cell of claim 29, wherein the promoter is an inducible promoter. The genetically modified cell of claim 31, wherein the inducible promoter is a modified T7 promoter. The genetically modified cell of claim 32, wherein the modified T7 promoter comprises a sequence selected from TAATACGACTCACTAATACTGAA (SEQ ID NO: 3), TAATACGACTCACTATTTCGGAA (SEQ ID NO: 4), TAATACGACTCTCTATAGG (SEQ ID NO: 5), TAATACGACTCACTATAGGAGAA (SEQ ID NO: 6), TAATACCACTCACTATAGGGAGA (SEQ ID NO: 7), TAATACAACTCACTATAGGGAGA (SEQ ID NO: 8), and

TAATACGTCTCACTATAGGGGAA (SEQ ID NO: 9). The genetically modified cell of claim 33, wherein the modified T7 promoter comprises the sequence TAATACGACTCTCTATAGG (SEQ ID NO: 5). The genetically modified cell of any one of claims 31-34, wherein the inducible promoter is an isopropyl P-d-1 -thiogalactopyranoside (IPTG)-inducible promoter. The genetically modified cell of claim 35, wherein the IPTG-inducible promoter is induced in the presence of 0.01 mM-3.0 mM IPTG. The genetically modified cell of claim 31, wherein the inducible promoter is a tetracycline (Tc)-inducible promoter. The genetically modified cell of claim 37, wherein the tetracycline (Tc)-inducible promoter comprises the sequence GTTGACACTCTATCATTGATAGAGTTATTTTACCACTC (SEQ ID NO: 69). The genetically modified cell of claim 37 or claim 38, wherein the tetracycline (Tc)- inducible promoter is induced in the presence of 1-1500 ng/ml anhydrotetracycline. The genetically modified cell of claim 39, wherein the tetracycline (Tc)-inducible promoter is induced in the presence of about 1000 ng/ml anhydrotetracycline. The genetically modified cell of claim 37 or claim 38, wherein the tetracycline (Tc)- inducible promoter is induced in the presence of 1-1500 ng/ml tetracycline. The genetically modified cell of claim 41, wherein the tetracycline (Tc)-inducible promoter is induced in the presence of about 1000 ng/ml tetracycline. The genetically modified cell of any one of claims 1-42, wherein the first polynucleotide and/or the additional polynucleotide(s) is integrated into a cellular chromosome. The genetically modified cell of claim 43, wherein the first polynucleotide is integrated into a cellular chromosome. The genetically modified cell of any one of claims 1-42, wherein the first polynucleotide and/or the additional polynucleotide(s) is present on a plasmid. The genetically modified cell of claim 45, wherein the additional polynucleotide(s) is present on a plasmid. The genetically modified cell of any one of claims 4-44, wherein the fusion polypeptide comprises the same toxin component as the toxin component of the endogenous TA module of said cell, and the activity of the endogenous TA module of said cell is eliminated by deletion of the gene encoding the endogenous TA module. The genetically modified cell of any one of claims 4-47, wherein the TA module is MazEF and the toxin component is MazF toxin, or a functional fragment or derivative thereof. The genetically modified cell of claim 48, wherein the MazF toxin, or the functional fragment or derivative thereof, comprises the amino acid sequence MVSRYVPDMGDLIWVDFDPTKGSEQAGHRPAVVLSPFMYNNKTGMCLCVPCTT QSKGYPFEVVLSGQERDGVALADQVKSIAWRARGATKKGTVAPEELQLIKAKIN VLIG (SEQ ID NO: 62). The genetically modified cell of any one of claims 7-49, wherein the first polynucleotide and/or the one or more additional polynucleotide(s) that are modified comprise the sequence Adenine-Cytosine-Adenine (ACA). The genetically modified cell of claim 50, wherein the ACA sequence(s) in the first polynucleotide and/or the one or more additional polynucleotide(s) is replaced such that the amino acid sequence(s) of the encoded polypeptide(s) is the same as when the ACA sequence(s) is not replaced. The genetically modified cell of any one of claims 6-51, wherein the biological substance is selected from biomass or its constituents, plasmid DNA, recombinant protein, peptide,

Ill amino acid, antibiotic, biosurfactant, biological fuel, organic acid, fatty acid, polyol, flavor, fragrance, polynucleotide, nucleotide, vitamin, pigment, sugar, polysaccharide, biopolymer, and plastic. The genetically modified cell of any one of claims 1-52, wherein the expression of the fusion polypeptide in said cell results in an increased efficiency of substrate utilization by the cell as compared to a control cell which does not express the fusion polypeptide. The genetically modified cell of claim 53, wherein the increased efficiency of substrate utilization is manifested in an increased production of biomass or a biological substance, or combination thereof, from the same amount of substrate utilized by the cell as compared to the control cell. The genetically modified cell of any one of claims 1-52, wherein the expression of the fusion polypeptide in said cell results in an increased production of biomass or a biological substance, or combination thereof, as compared to a control cell which does not express the fusion polypeptide. The genetically modified cell of any one of claims 1-52, wherein the expression of the fusion polypeptide in said cell results in a slower rate of substrate utilization by the cell as compared to a control cell which does not express the fusion polypeptide. The genetically modified cell of claims 56, wherein the slower rate of substrate utilization is manifested in an increased duration of cellular growth. The genetically modified cell of any one of claims 1-57, wherein the fusion polypeptide possesses an enzymatic activity. A genetically modified E. coll cell comprising a first polynucleotide encoding a fusion polypeptide comprising MazF toxin, or a functional fragment or derivative thereof, operably linked to a signal peptide pelB or TorA, or a functional fragment or derivative thereof, wherein expression of the fusion polypeptide is under the control of a modified T7 promoter or a tetracycline (Tc)-inducible promoter, and wherein the endogenous toxinantitoxin (TA) module MazEF has been deleted. The genetically modified E. coli cell of claim 59, wherein the MazF toxin, or the functional fragment or derivative thereof, comprises the amino acid sequence MVSRYVPDMGDLIWVDFDPTKGSEQAGHRPAVVLSPFMYNNKTGMCLCVPCTT QSKGYPFEVVLSGQERDGVALADQVKSIAWRARGATKKGTVAPEELQLIKAKIN VLIG (SEQ ID NO: 62). The genetically modified E. coli cell of claim 59 or claim 60, wherein the PelB signal peptide, or the functional fragment or derivative thereof, comprises the amino acid sequence MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 1). The genetically modified E. coli cell of claim 59 or claim 60, wherein the TorA signal peptide, or the functional fragment or derivative thereof, comprises the amino acid sequence MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 2). The genetically modified E. coli cell of any one of claims 59-62, wherein the modified T7 promoter comprises a sequence selected from TAATACGACTCACTAATACTGAA (SEQ ID NO: 3), TAATACGACTCACTATTTCGGAA (SEQ ID NO: 4),

TAATACGACTCTCTATAGG (SEQ ID NO: 5), TAATACGACTCACTATAGGAGAA (SEQ ID NO: 6), TAATACCACTCACTATAGGGAGA (SEQ ID NO: 7), TAATACAACTCACTATAGGGAGA (SEQ ID NO: 8), and

TAATACGTCTCACTATAGGGGAA (SEQ ID NO: 9). The genetically modified E. coli cell of claim 63, wherein the modified T7 promoter comprises the sequence TAATACGACTCTCTATAGG (SEQ ID NO: 5). The genetically modified E. coli cell of any one of claims 59-64, wherein the tetracycline

(Tc)-inducible promoter comprises the sequence

GTTGACACTCTATCATTGATAGAGTTATTTTACCACTC (SEQ ID NO: 69). The genetically modified E. coli cell of any one of claims 59-65, wherein the first polynucleotide is integrated into a cellular chromosome. The genetically modified E. coli cell of any one of claims 59-66, further comprising one or more additional polynucleotides, said additional polynucleotide(s) encoding at least one polypeptide which is a biological substance or which participates in the production of the biological substance, wherein the first polynucleotide encoding the fusion polypeptide and/or the additional polynucleotide(s) encoding the at least one polypeptide is modified to replace one or more ACA nucleotide sequences in the corresponding mRNA(s). The genetically modified E. coli cell of claim 67, wherein the ACA sequence(s) in the first polynucleotide and/or the one or more additional polynucleotide(s) is replaced such that the amino acid sequence(s) of the encoded polypeptide(s) is the same as when the ACA sequence(s) is not replaced. The genetically modified E. coli cell of claim 67 or claim 68, wherein the biological substance is selected from biomass or its constituents, plasmid DNA, recombinant protein, peptide, amino acid, antibiotic, biosurfactant, biological fuel, organic acid, fatty acid, polyol, flavor, fragrance, polynucleotide, nucleotide, vitamin, pigment, sugar, polysaccharide, biopolymer, and plastic. A polynucleotide molecule comprising a polynucleotide sequence encoding a fusion polypeptide comprising a global metabolic regulator, or a component or functional fragment or derivative thereof, operably linked to a signal peptide which enables secretion of the fusion polypeptide to the space outside of the cytoplasmic membrane of a genetically modified cell upon expression of the fusion polypeptide is said cell. The polynucleotide molecule of claim 70, wherein the expression of the fusion polypeptide in the genetically modified cell does not fully inhibit growth of said cell. The polynucleotide molecule of claim 71, wherein the growth rate of said genetically modified cell during the expression of the fusion polypeptide is higher than 0. The polynucleotide molecule of claim 70, wherein the global metabolic regulator operably linked to the signal peptide is a toxin component of a toxin and antitoxin (TA) module, or a functional fragment or derivative thereof. The polynucleotide molecule of claim 73, wherein the fusion polypeptide comprises the same toxin component, or the functional fragment or derivative thereof, as the toxin component of an endogenous TA module of said cell. The polynucleotide molecule of any one of claims 70-74, further comprising one or more additional polynucleotide sequences, said additional polynucleotide sequence(s) encoding at least one polypeptide which is a biological substance or which participates in the production of the biological substance. The polynucleotide molecule of claim 75, wherein the polynucleotide sequence encoding the fusion polypeptide and/or the one or more additional polynucleotide sequence(s) encoding the at least one polypeptide is modified to replace one or more nucleotide sequences in a corresponding mRNA(s) that are recognizable by said toxin component, or the functional fragment or derivative thereof, so that such mRNA(s) becomes resistant to the destruction by said toxin component. The polynucleotide molecule of claims 70-76, wherein the genetically modified cell is a microbial cell. The polynucleotide molecule of claim 77, wherein the microbial cell is a prokaryotic cell. The polynucleotide molecule of claim 78, wherein the prokaryotic cell is a bacterial cell. The polynucleotide molecule of claim 78 or claim 79 wherein the prokaryotic cell is from a genus selected from Nocardia, Acetobacter, Bacillus, Clostridium, Corynebacterium, Erwinia, Escherichia, Gluconacetobacter, Gluconobacter, Klebsiella, Lactococcus, Lactobacillus, Mannheimia, Propionibacterium, Pseudomonas, Ralstonia, Saccharopolyspora, Salmonella, Serratia, Streptomyces, Xanthomonas, and Zymomonas. The polynucleotide molecule of claim 80, wherein the prokaryotic cell is from a species selected from Acetobacter aceti, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus subtilis, Clostridium acetobutylicum, Clostridium butyricum, Clostridium thermoaceticum, Clostridium tyrobutyricum, Corynebacterium glutamicum, Erwinia citreus, Erwinia herbicola, Gluconacetobacter hansenii, Gluconobacter oxydans, Klebsiella oxytoca, Lactobacillus delbrueckii, Lactobacillus rhamnosus. Mannheimia succinicip-roducens. Nocardia lactamdurans, Propionibacterium shermanii. Pseudomonas denilrificans. Ralstonia eulropha. Saccharopolyspora erylhrea. Saccharopolyspora spinosa. Serratia marcescens. Streptomyces clavuligerus, Streptomyces griseus. Streptomyces Hvidans. Streptomyces roseosporus. Xanthomonas campeslris. Zymomonas mobilis. Escherichia coli, Lactococcus lactis, Bacillus cereus. Salmonella lyphimurium. and Pseudomonas fluorescens. The polynucleotide molecule of claim 81, wherein the prokaryotic cell is from a species selected from Escherichia coli, Lactococcus lactis, Bacillus subtilis, Bacillus cereus, Salmonella typhimurium, and Pseudomonas fluorescens. The polynucleotide molecule of claim 82, wherein the prokaryotic cell is from the species Escherichia coli. The polynucleotide molecule of any one of claims 77-83, wherein the signal peptide enables secretion of the fusion polypeptide to the periplasm of the microbial cell. The polynucleotide molecule of any one of claims 77-84, wherein the signal peptide enables secretion of the fusion polypeptide to the extracellular space of the microbial cell. The polynucleotide molecule of any one of claims 73-85, wherein the signal peptide is a Sec-dependent signal peptide, or a twin-arginine translocation (TAT)-dependent signal peptide. The polynucleotide molecule of claim 86, wherein the signal peptide is a Sec-dependent signal peptide selected from LamB, LTB, MalE, OmpA, PelB, PhoA, SpA, PhoE, OmpT, OmpF, and functional fragments or derivatives thereof. The polynucleotide molecule of claim 87, wherein the signal peptide is a PelB signal peptide or a functional fragment or derivative thereof. The polynucleotide molecule of claim 88, wherein the PelB signal peptide, or the functional fragment or derivative thereof, comprises the amino acid sequence MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 1). The polynucleotide molecule of claim 86, wherein the signal peptide is a TAT-dependent signal peptide selected from TorA, Tap, and functional fragments or derivatives thereof. The polynucleotide molecule of claim 90, wherein the signal peptide is a TorA signal peptide or a functional fragment or derivative thereof. The polynucleotide molecule of claim 91, wherein the TorA signal peptide, or the functional fragment or derivative thereof, comprises the amino acid sequence MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 2). The polynucleotide molecule of any one of claims 70-92, wherein the polynucleotide sequence encoding the fusion polypeptide is operably linked to a promotor. The polynucleotide molecule of claim 93, wherein the promoter is a constitutive promoter. The polynucleotide molecule of claim 93, wherein the promoter is an inducible promoter. The polynucleotide molecule of claim 95, wherein the inducible promoter is a modified T7 promoter. The polynucleotide molecule of claim 96, wherein the modified T7 promoter comprises a sequence selected from TAATACGACTCACTAATACTGAA (SEQ ID NO: 3), TAATACGACTCACTATTTCGGAA (SEQ ID NO: 4), TAATACGACTCTCTATAGG (SEQ ID NO: 5), TAATACGACTCACTATAGGAGAA (SEQ ID NO: 6), TAATACCACTCACTATAGGGAGA (SEQ ID NO: 7), TAATACAACTCACTATAGGGAGA (SEQ ID NO: 8), and

TAATACGTCTCACTATAGGGGAA (SEQ ID NO: 9). The polynucleotide molecule of claim 97, wherein the modified T7 promoter comprises the sequence TAATACGACTCTCTATAGG (SEQ ID NO: 5). The genetically modified cell of any one of claims 95-98, wherein the inducible promoter is an isopropyl P-d-1 -thiogalactopyranoside (IPTG)-inducible promoter. . The polynucleotide molecule of claim 95, wherein the inducible promoter is a tetracycline (Tc)-inducible promoter. . The polynucleotide molecule of claim 100, wherein the tetracycline (Tc)-inducible promoter comprises the sequence

GTTGACACTCTATCATTGATAGAGTTATTTTACCACTC (SEQ ID NO: 69) . The polynucleotide molecule of any one of claims 70-101, wherein the fusion polypeptide comprises the same toxin component, or the functional fragment or derivative thereof, as the toxin component of the endogenous TA module of said cell. . The polynucleotide molecule of any one of claims 70-102, wherein the TA module is MazEF and the toxin component is MazF toxin, or a functional fragment or derivative thereof. . The polynucleotide molecule of claim 103, wherein the MazF toxin, or the functional fragment or derivative thereof, comprises the amino acid sequence MVSRYVPDMGDLIWVDFDPTKGSEQAGHRPAVVLSPFMYNNKTGMCLCVPCTT QSKGYPFEVVLSGQERDGVALADQVKSIAWRARGATKKGTVAPEELQLIKAKIN VLIG (SEQ ID NO: 62). . The polynucleotide molecule of any one of claims 76-104, wherein the one or more nucleotide sequences in the corresponding mRNA(s) that are recognizable by the toxin component of the TA module, or the functional fragment or derivative thereof, comprise the sequence Adenine-Cytosine- Adenine (AC A). . The polynucleotide molecule of claim 105, wherein the ACA sequence(s) in the corresponding mRNA(s) is replaced such that the amino acid sequence(s) of the encoded polypeptide(s) is the same as when the ACA sequence(s) is not replaced.

. The polynucleotide molecule of any one of claims 75-106, wherein the biological substance is selected from biomass or its constituents, plasmid DNA, recombinant protein, peptide, amino acid, antibiotic, biosurfactant, biological fuel, organic acid, fatty acid, polyol, flavor, fragrance, polynucleotide, nucleotide, vitamin, pigment, sugar, polysaccharide, biopolymer, and plastic. . A polynucleotide molecule comprising a polynucleotide sequence encoding a fusion polypeptide comprising MazF toxin, or a functional fragment or derivative thereof, operably linked to a signal peptide pelB or TorA, or a functional fragment or derivative thereof, wherein the polynucleotide sequence encoding the fusion polypeptide is optionally operably linked to a modified T7 promoter or a tetracycline (Tc)-inducible promoter. . The polynucleotide molecule of claim 108, wherein the MazF toxin, or the functional fragment or derivative thereof, comprises the amino acid sequence MVSRYVPDMGDLIWVDFDPTKGSEQAGHRPAVVLSPFMYNNKTGMCLCVPCTT QSKGYPFEVVLSGQERDGVALADQVKSIAWRARGATKKGTVAPEELQLIKAKIN VLIG (SEQ ID NO: 62). . The polynucleotide molecule of claim 108 or claim 109, wherein the PelB signal peptide, or the functional fragment or derivative thereof, comprises the amino acid sequence MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 1). . The polynucleotide molecule of claim 108 or claim 109, wherein the TorA signal peptide, or the functional fragment or derivative thereof, comprises the amino acid sequence MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 2). . The polynucleotide molecule of any one of claims 108-111, wherein the modified T7 promoter comprises a sequence selected from TAATACGACTCACTAATACTGAA (SEQ ID NO: 3), TAATACGACTCACTATTTCGGAA (SEQ ID NO: 4),

TAATACGACTCTCTATAGG (SEQ ID NO: 5), TAATACGACTCACTATAGGAGAA (SEQ ID NO: 6), TAATACCACTCACTATAGGGAGA (SEQ ID NO: 7), TAATACAACTCACTATAGGGAGA (SEQ ID NO: 8), and

TAATACGTCTCACTATAGGGGAA (SEQ ID NO: 9).

. The polynucleotide molecule of claim 112, wherein the modified T7 promoter comprises the sequence TAATACGACTCTCTATAGG (SEQ ID NO: 5). . The polynucleotide molecule of any one of claims 108-113, wherein the tetracycline

(Tc)-inducible promoter comprises the sequence

GTTGACACTCTATCATTGATAGAGTTATTTTACCACTC (SEQ ID NO: 69). . The polynucleotide molecule of any one of claims 108-114, further comprising one or more additional polynucleotide sequences, said additional polynucleotide sequence(s) encoding at least one polypeptide which is a biological substance or which participates in the production of the biological substance, wherein the polynucleotide sequence encoding the fusion polypeptide and/or the additional polynucleotide sequence(s) encoding the at least one polypeptide is modified to replace one or more ACA nucleotide sequences in a corresponding mRNA(s). . The polynucleotide molecule of claim 115, wherein the ACA sequence(s) in the corresponding mRNA(s) is replaced such that the amino acid sequence(s) of the encoded polypeptide(s) is the same as when the ACA sequence(s) is not replaced. . The polynucleotide molecule of claim 115 or claim 116, wherein the biological substance is selected from biomass or its constituents, plasmid DNA, recombinant protein, peptide, amino acid, antibiotic, biosurfactant, biological fuel, organic acid, fatty acid, polyol, flavor, fragrance, polynucleotide, nucleotide, vitamin, pigment, sugar, polysaccharide, biopolymer, and plastic. . The polynucleotide molecule of any one of claims 70-117, wherein the fusion polypeptide possesses an enzymatic activity. . A recombinant construct comprising the polynucleotide molecule of any one of claims 70-118. . The recombinant construct of claim 119, wherein the construct is a plasmid.

. A polynucleotide molecule comprising a modified T7 promoter comprising a sequence selected from TAATACGACTCACTAATACTGAA (SEQ ID NO: 3), TAATACGACTCACTATTTCGGAA (SEQ ID NO: 4), TAATACGACTCTCTATAGG (SEQ ID NO: 5), TAATACGACTCACTATAGGAGAA (SEQ ID NO: 6), TAATACCACTCACTATAGGGAGA (SEQ ID NO: 7), TAATACAACTCACTATAGGGAGA (SEQ ID NO: 8), and TAATACGTCTCACTATAGGGGAA (SEQ ID NO: 9). . The polynucleotide molecule of claim 121, wherein the modified T7 promoter comprises the sequence TAATACGACTCTCTATAGG (SEQ ID NO: 5). . A recombinant construct comprising the polynucleotide molecule of claim 118. . A method of producing a biological substance, comprising culturing the genetically modified cell of any one of claims 6-58 and 67-69 under conditions suitable for producing the biological substance, and optionally purifying the biological substance. . A method of increasing efficacy of substrate utilization by a cell, said method comprising genetically modifying said cell by introducing the polynucleotide molecule of any one of claims 70-118 or the recombinant construct of claim 119 into the cell. . A method of increasing production of biomass or a biological substance, or a combination thereof, by a cell, said method comprising genetically modifying said cell by introducing the polynucleotide molecule of any one of claims 70-118 or the recombinant construct of claim 119 into the cell.