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WO2025216711A1 - Procédé de préparation de propane-1,3-diol - Google Patents

Procédé de préparation de propane-1,3-diol

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Publication number
WO2025216711A1
WO2025216711A1 PCT/SG2025/050246 SG2025050246W WO2025216711A1 WO 2025216711 A1 WO2025216711 A1 WO 2025216711A1 SG 2025050246 W SG2025050246 W SG 2025050246W WO 2025216711 A1 WO2025216711 A1 WO 2025216711A1
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WIPO (PCT)
Prior art keywords
diolivorans
glycerol
cells
coli
propanediol
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Pending
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PCT/SG2025/050246
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English (en)
Inventor
Hong Li
Bo XUE
Wen Shan YEW
Xiao Wang
Ye Qing TENG
Guan Qiang TENG
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Hh Chemical Pte Ltd
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Hh Chemical Pte Ltd
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Publication of WO2025216711A1 publication Critical patent/WO2025216711A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01202—1,3-Propanediol dehydrogenase (1.1.1.202)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/0103—Glycerol dehydratase (4.2.1.30)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • the present invention relates, in general terms, to methods of preparing 1,3 -propanediol.
  • the specification teaches a method of preparing 1,3-propanediol from glycerol using bacteria.
  • Synthetic polymers arc the cornerstone of modem life, underpinning a multitude of essential products such as plastics, fibers, and adhesives. Their omnipresent and versatile nature position them as indispensable elements across various industries and everyday applications. However, many synthetic polymers are made from fossil fuels, which are not sustainable. Recently, there has been more focus on developing biotechnological methods to produce polymers from renewable resources.
  • Polytrimethylene terephthalate (PTT) is a polymer which finds extensive use in the food, pharmaceuticals, cosmetics and lubricant industries.
  • PTT is synthesised from a 1,3-propanediol (1,3-PD) precursor, which can be produced biologically from a variety of carbon sources, among which glycerol is an attractive feedstock due to the relatively simple metabolism pathway from glycerol to 1,3-PD, and the abundance of low-cost crude glycerol as a byproduct from the biofuel industry.
  • glycerol is an attractive feedstock due to the relatively simple metabolism pathway from glycerol to 1,3-PD, and the abundance of low-cost crude glycerol as a byproduct from the biofuel industry.
  • glycerol is an attractive feedstock due to the relatively simple metabolism pathway from glycerol to 1,3-PD, and the abundance of low-cost crude glycerol as a byproduct from the biofuel industry.
  • glycerol is an attractive feedstock due to the relatively simple metabolism pathway from glycerol to 1,3-PD
  • the abundance of low-cost crude glycerol as a
  • a method of preparing 1,3-propanediol from glycerol comprising: a) cultivating Lentilactobacillus diolivorans cells in a cell culture in the presence of glycerol to produce 3-hydroxypropionaldehyde (3-HPA) and/or 1,3-propanediol (1,3-PD), wherein the Lentilactobacillus diolivorans cells arc genetically engineered to overexpress a glycerol transporter; and b) cultivating Escherichia coli cells in cell culture media from step a) to produce 1,3 -propanediol, wherein the Escherichia coli cells are genetically engineered to overexpress a 1 ,3-propanediol oxidoreductase.
  • Disclosed herein is a method of preparing 1,3-propanediol from glycerol, the method comprising cultivating Lentilactobacillus diolivorans cells and Escherichia coli cells in the presence of glycerol, wherein the L. diolivorans is genetically engineered to overexpress a glycerol transporter, and wherein the E. coli is genetically engineered to overexpress a 1,3- propanediol oxidoreductase.
  • kits for biological production of 1,3-propanediol from glycerol comprising Lentilactobacillus diolivorans cells genetically engineered to overexpress a glycerol transporter, and Escherichia coli cells genetically engineered to overexpress a 1,3- propanediol oxidoreductase.
  • FIG. 1 Plasmid maps for gene overexpression in L. diolivorans.
  • Figure 2 Modular whole-cell biotransformation for 1,3-PD production using engineered L. diolivorans and E. coli recombinants, (i) Glycerol metobolic pathway in gene ovcrcxprcssionL. diolivorans recombinant, (ii) Bioconversion of 3-HPA into 1,3-PD in 1,3- PD oxidoreductase overexpression E. coli recombinant.
  • FIG. 3 The production of 3-HPA and 1 ,3-PD in different L. diolivorans strains and optimization fermentation condition for the first step whole-cell biotransformation with LMG199668//? ⁇ 7MF.
  • LMG19668/EV is a control strain which was transformed with empty vector
  • FIG. 11 development SDS-PAGE analysis of 1,3-PD oxidoreductases (PDORs) from different microorganisms expressed in E. coli BL21 (DE3).
  • Lane M protein marker
  • Lane 1 PDOR from Clostridium butyricum DSM5478
  • Lane 2 PDOR from Lentilactobacillus diolivorans
  • Lane 3 PDOR from Clostridium butyricum VPI1718
  • Lane 4 PDOR from Clostridium pasteurianum
  • Lane 5 PDOR from Klebsiella pneumoniae
  • Lane 6 PDOR from Citrobacter freundii.
  • FIG. 5 The production of 3-HPA and 1,3-PD in the two modules and the reuse of the cells, (a) The production of 3-HPA and 1,3-PD after the first step whole-cell biotransformation with L. diolivorans recombinant and after second step whole-cell biotransformation with E. coli recombinants, (b) Test reuse of the cells of L. diolivorans recombinant and E. coli recombinant for 3-HPA and 1,3-PD production.
  • FIG. 6 The HPLC chromatograms of culture media from the first step and second step whole-cell biotransformation, (a) The HPLC chromatogram of the first step whole-cell biotransformation with LMG196684? ⁇ 7MF. (b) The HPLC chromatogram of the second step whole-cell biotransformation with BL-dhaT.
  • Module I development 1,3-PD production by stepwise engineered L. diolivorans strains and fermentation optimization of the selected optimal producer, (a) 1 ,3-PD production by stepwise engineered L. diolivorans strains, (b) Enhanced 1,3-PD production by the selected optimal strain (Lac/4) after response surface optimization.
  • Figure 8. The concept of modular co-cultivation system, in which Module I consists of an L. diolivorans recombinant and Module II consists of an E. coll recombinant.
  • Figure 9 Optimization of co-cultivation systems: combining fixed concentration of optimal L. diolivorans strain from Module I (5 g/L) with varying ratios of engineered E. coli strains from Module II. Co-cultivation performance with the optimal strain from Module I (Lac/4, 5 g/L) and engineered E. coli from Module II, where E. coli comprises: (a) 25% of total biomass, (b) 50% of total biomass, (c) 75% of total biomass, (d) Enhanced 1,.3-PD production and reduced 3-HPA accumulation at 1:1 strain ratio compared to wild-type LMG19668.
  • the L. diolivorans is genetically engineered to overexpress a glycerol transporter to increase glycerol uptake, and may also be engineered to overexpress a glycerol dehydratase with its activation factor, and/or a 1,3-propanediol oxidoreductase (PDOR).
  • PDOR 1,3-propanediol oxidoreductase
  • coli is engineered to overexpress a 1,3-propanediol oxidoreductase (PDOR).
  • PDOR 1,3-propanediol oxidoreductase
  • Glycerol dehydratase converts glycerol to an intermediate compound 3-hydroxypropionaldchydc (3-HPA), while 1,3-propancdiol oxidoreductase converts 3-HPA to 1,3-PD ( Figure 2).
  • L. diolivorans natively express glycerol metabolic pathway enzymes
  • conversion of glycerol to 1,3-PD can be inefficient in these strains and limited by accumulation of toxic 3-HPA in the culture.
  • the inventors found that engineering L. diolivorans to overexpress a glycerol transporter, glycerol dehydratase and/or 1,3- propanediol oxidoreductase can improve glycerol biotransformation.
  • engineered E. coli strains that overexpress 1,3-propanediol oxidoreductase can be used in conjunction with the engineered L. diolivorans to efficiently convert the 3-HPA intermediate to further increase the yield of 1 ,3-PD.
  • the inventors show that the engineered L. diolivorans and E. colt cells may be cultured sequentially or together.
  • the L. diolivorans may be cultured first in the presence of glycerol to produce a conditioned culture containing 3-HPA and/or 1,3-PD, and the E. coli is then grown in the conditioned culture to produce 1 ,3-PD.
  • the L. diolivorans and the E. coli may co-cultured in the presence of glycerol to produce 1,3- PD.
  • the combination of increased glycerol uptake and glycerol metabolism in the engineered L. diolivorans and the heterologous 1,3-PD production pathway in the engineered E. coli creates a synergistic system for high-yield 1,3-PD biosynthesis from glycerol.
  • the inventors were able to achieve significant improvements in 1,3-PD yields over current bioproduction methods, with yields exceeding 28 g/L.
  • glycerol transporter refers to a membrane-associated protein that facilitates glycerol uptake into a cell.
  • the transporter may facilitate unidirectional transport of glycerol into the cell, or bidirectional transport of glycerol into and out of the cell.
  • glycerol dehydratase refers to an enzyme which catalyses the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA).
  • the enzyme may be a multimeric protein containing, for example, two, three or more subunits.
  • Glycerol dehydratases may be dependent on adenosylcobalamin (vitamin B 12) as a cofactor, or can exist in B 12- independent forms. Glycerol dehydratases may become inactivated over the course of catalysis, for example by the glycerol substrate or by exposure to oxygen. Co-expression of an “activating factor” or “activase” can help to restore enzyme activity. The gene for activating factors are frequently found alongside the gene for the glycerol dehydratase.
  • 1,3-propanediol oxidoreductase refers to an enzyme which catalyses the reduction of 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD), typically converting it.
  • the enzyme is also known as 1,3-propanediol dehydrogenase.
  • overexpression as used herein with respect to a host cell, specifically a recombinant host cell, is intended to encompass increasing the expression of a polypeptide or protein (such as enzymes used in the biotransformation process) to a level greater than the cell normally produces. It is intended that the term encompass overexpression of homologous (endogenous), as well as heterologous sequences or proteins, specifically aiming at the increase of the productivity of the cell to produce a product, e.g., at least 1.5- fold, preferably at least 2-fold, more preferred at least 3-fold as compared to a wild-type host cell of the same type.
  • “Expression vectors” or “vectors” used herein are DNA sequences that are required for the transcription of cloned recombinant nucleotide sequences, i.e. of recombinant genes and the translation of their mRNA in a suitable host organism.
  • Such expression vectors usually comprise an origin for autonomous replication in the host cells, selectable markers (e.g. an amino acid synthesis gene or a gene conferring resistance to antibiotics such as erythromycin, chloramphenicol, zeocin, kanamycin, G418 or hygromycin), a number of restriction enzyme cleavage sites, a suitable promoter sequence and a transcription terminator, which components are operably linked together.
  • selectable markers e.g. an amino acid synthesis gene or a gene conferring resistance to antibiotics such as erythromycin, chloramphenicol, zeocin, kanamycin, G418 or hygromycin
  • a number of restriction enzyme cleavage sites a
  • genetically engineered refers to a recombinant organism that is used for producing a fermentation product.
  • the organism is typically a production cell line engineered to improve a production process or to enable the production of a new product.
  • the term is understood in contrast to the “wild-type” organism, which is typically not genetically engineered.
  • a “recombinant” as used herein means “being prepared by or the result of genetic engineering”.
  • a “recombinant microorganism” comprises at least one “recombinant nucleic acid”.
  • a recombinant microorganism may be a mutant produced by a suitable method mutagenesis, and/or specifically comprises an expression vector or cloning vector, or it has been genetically engineered to contain a recombinant nucleic acid sequence or recombinant metabolic pathway to produce cell metabolites at high yield or to produce novel cell metabolites which the wild-type cell (before recombination) would not produce in significant amounts.
  • cell line shall refer to an established clone of a particular cell type that has acquired the ability to proliferate over a prolonged period of time.
  • host cell line refers to a cell line as used for expressing an endogenous or recombinant gene or products of a metabolic pathway to produce polypeptides or cell metabolites mediated by such polypeptides.
  • a “production host cell line” or “production cell line” is commonly understood to be a cell line ready-to-use for cultivation in a bioreactor to obtain the product of a production process.
  • the applicable production cell line may produce a number of useful intermediates and products by fermenting a low molecular weight sugar produced by saccharifying the treated biomass materials.
  • the applicable production cell line may also produce the useful intermediates and products while saccharifying the treated biomass materials by producing the necessary enzymes. For example, fermentation or other bioprocesses can produce alcohols, organic acids, hydrocarbons, hydrogen, proteins or mixtures of any of these materials.
  • the recombinant nucleic acids or organisms as referred to herein may be produced by recombination techniques well known to a person skilled in the art.
  • conventional methods of molecular' biology, microbiology, and recombinant DNA techniques within the skill of the art may be employed. Such techniques are explained in detail e.g., in Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory Manual (1982).
  • cell culture or “culturing” or “cultivation” as used herein refers to the maintenance of the bacterial cells in an artificial, e.g., an in vitro environment, under conditions favouring growth, differentiation, or continued viability, in an active or quiescent state, of the cells, specifically in a controlled biorcactor according to methods known in the industry.
  • the cells When culturing a cell culture using appropriate culture media, the cells are brought into contact with the media in a culture vessel or with substrate under conditions suitable to support culturing the cells in the cell culture.
  • a culture medium is provided that can be used for the growth of cells. Standard cell culture techniques are well- known in the art.
  • conditioned culture herein refers to a culture that has been exposed to cells for a period of time, such as hours, days or longer, and which contains metabolites and other metabolic by-products produced by the cells.
  • Consditioned medium refers to culture medium isolated from a conditioned culture.
  • sequence identity indicates that two or more nucleotide sequences have (to a certain degree, up to 100%) the same or conserved base pairs at a corresponding position.
  • Percent (%) identity with respect to the nucleotide sequence of a gene or genome is defined as the percentage of nucleotides in a candidate DNA sequence that is identical with the nucleotides in the DNA sequence to which the candidate sequence shall be compared, after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • an agent includes a plurality of agents, including mixtures thereof.
  • L. diolivorans that can be used include wild-type and deposited strains such as LMG19668, G77, DSM14421, DSM14424, DSM33056, DSM33057, DSM33058, DSM33059, DSM33060, or any derivatives thereof. These strains can be obtained from, e.g., the Belgian Coordinated Collections of Microorganisms (BCCM), or the Leibniz Institute DSMZ-Gcrman Collection of Microorganisms and Cell Cultures.
  • the L. diolivorans cells are L. diolivorans LMG199668 orL. diolivorans DSM14421. These strains possess a native pathway for 1,3-PD production, can directly utilise crude glycerol, have a Generally Regarded as Safe (GRAS) status, and have high stress tolerance during culture, and are advantageous for the methods herein.
  • GRAS Generally Regarded as Safe
  • L. diolivorans may be cultivated in any suitable media known in the ail for L. diolivorans culture.
  • the medium may be a defined medium or a complex medium.
  • media commonly used for Lactobacillus culture such as MRS medium, M17 medium or clostridial medium, may be used for L. diolivorans cultivation.
  • media derived from agricultural or industrial byproducts such as whey-based formulations and formulations containing hydrolysates of starchy plant material or hydrolysates of lignocellulosic plant material.
  • the L. diolivorans medium contains glycerol as raw material for bioconversion to 3-HPA and 1,3-PD.
  • the medium may contain one or more additional carbon sources such as glucose, xylose, arabinose, fructose, sucrose, lactose or others to support growth of the bacteria.
  • the L. diolivorans cells are cultivated in the presence of about 0.5% w/v to about 3% w/v glycerol, such as about 0.5% w/v, about 1% w/v, about 1.5% w/v, about 2% w/v, about 2.5% w/v, or about 3% w/v glycerol.
  • the inventors have found that glycerol concentrations higher than about 3% w/v can lead to reduced yields of both 3-HPA and 1,3- PD.
  • the glycerol may be crude or refined glycerol.
  • Crude glycerol is generated by a number of commercial and experimental processes, including saponification, e.g., hydrolysis of triacylglyccrol to produce fatty acids and glycerol, and biodiesel production, in which transesterification of fats and oils occurs to produce methyl esters and a glycerol byproduct.
  • Crude glycerol typically contains contaminants and/or impurities. The removal of these contaminants and/or impurities using processes known in the art produces refined glycerol.
  • Refined glycerol may be sold as a product that is, e.g., at least 99.5% pure.
  • the L. diolivorans medium may be supplemented with vitamin B12.
  • Vitamin B 12 is a cofactor for B12-dcpcndcnt glycerol dehydratases, and although the vitamin is present in most microbial complex media, B 12 supplementation can improve the efficiency of glycerol bioconversion, in particular where the L. diolivorans is engineered to overexpress a B 12- dependent glycerol dehydratase.
  • vitamin B 12 is provided at a concentration of about 5 mg/ml in the L. diolivorans medium.
  • L. diolivorans may be cultivated at a temperature in the range of about 25°C to about 40°C. Different culture temperatures may be used for protein expression and biotransformation.
  • the L. diolivorans may be cultured at an initial lower temperature (such as temperatures below 30°C) for transporter or enzyme overexpression, and the culture temperature is then raised for more efficient biotran formation.
  • the L. diolivorans is cultivated at a temperature in the range of about 30°C to about 37°C for biotransformation.
  • the L. diolivorans is cultivated at a temperature of about 30°C for biotransformation.
  • L. diolivorans is a facultative anaerobe and may be cultured under aerobic or anaerobic conditions. In one embodiment, the L. diolivorans is cultured anaerobically when in monoculture and aerobically when in co-culture with E. coli. In one embodiment, the L. diolivorans is cultured anaerobically both in monoculture and in co-culture with the E. coli. In one embodiment, the L. diolivorans is cultured anaerobically during the growth phase and aerobically during the production phase. In one embodiment, the L. diolivorans is cultured anaerobically for both the growth phase and the production phase.
  • E. coli strains suitable for industrial production are well known in the art. These include E. coli K-12 derivatives such as MG1655 and W3110, B strains such as BL21 and C41, and E. coli W.
  • E. coli may be cultivated in any suitable culture media, including both defined and complex media.
  • Complex media may include, for example, LB broth, Tryptic Soy Broth, Terrific Broth, 2xYT, and the like.
  • media derived from agricultural or industrial byproducts such as formulations containing hydrolysates of starchy plant material or hydrolysates of lignocellulosic plant material.
  • E. coli may be cultivated at a temperature in the range of about 15°C to about 40°C. Different culture temperatures may be used for protein expression and biotransformation. For example, the E. coli may be cultured at an initial lower temperature (such as temperatures below 20°C) for transporter or enzyme overexpression, and a higher culture temperature is then used for biotransformation. In some embodiments, the E. coli is cultivated in the range of about 30°C to about 37°C for biotransformation. In one embodiment, the E. coli is cultivated al a temperature of about 30°C for biotransformation.
  • E. coli is a facultative anaerobe and may be cultured under aerobic or anaerobic conditions. In one embodiment, the E. coli is cultured aerobically when in monoculture and anaerobically when in co-culture with L. diolivorans. In one embodiment, the E. coli is cultured aerobically both in monoculture and in co-culture with the L. diolivorans. In one embodiment, the E. coli is cultured aerobically during the growth phase and anaerobically during the production phase. In one embodiment, the E. coli is cultured aerobically during both the growth and the production phases.
  • the L. diolivorans is engineered to overexpress a glycerol transporter to increase uptake of glycerol for biotransformation.
  • the glycerol transporter may be a bacterial glycerol transporter.
  • the glycerol transporter may, for example, be derived from Lentilactobacillus diolivorans, Escherichia coli or Bacillus suhtilis.
  • the glycerol transporter is derived from L. diolivorans.
  • the L. diolivorans is engineered to overexpress a native glycerol transporter.
  • the L. diolivorans is engineered to overexpress a glycerol transporter derived from L. diolivorans DSM14421 or LMG19668.
  • the glycerol transporter comprises an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity) to an amino acid sequence in SEQ ID NO: 1.
  • the overexpression of the glycerol transporter may increase uptake of glycerol into the engineered L. diolivorans cells compared to wild-type L. diolivorans.
  • the genetically engineered L. diolivorans cells are characterised by an increased uptake of glycerol compared to wild-type L. diolivorans cells.
  • the engineered L. diolivorans may exhibit an increase in glycerol uptake that is at least 10%, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, or more than 5-fold, compared to the wild-type L. diolivorans cells from which they were engineered.
  • the L. diolivorans is engineered to overexpress a glycerol dehydratase and a glycerol dehydratase activation factor to increase conversion of glycerol to 3-HPA.
  • the glycerol dehydratase may be a bacterial glycerol dehydratase.
  • the glycerol dehydratase is a bacterial B 12-dependent glycerol dehydratase. In one embodiment, the glycerol dehydratase is a B 12-dependent glycerol dehydratase derived from Lentilactobacillus diolivorans .
  • the B12-dcpcndcnt glycerol dehydratase comprises a polypeptide comprising an amino acid sequence having at least 70%> sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity) to an amino acid sequence in SEQ ID NO: 3, 5 or 7.
  • the glycerol dehydratase is a bacterial B12-independent glycerol dehydratase.
  • the glycerol dehydratase is a B12-independent glycerol dehydratase derived from Clostridium butyricum or Lentilactobacillus diolivorans.
  • the expression of a B12-independent glycerol dehydratase and its activation factor can be advantageous in enabling efficient glycerol-to-3-HPA conversion without requiring vitamin B 12 supplementation during culture.
  • the B 12-independent glycerol dehydratase comprises a polypeptide comprising an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity) to an amino acid sequence in SEQ ID NO: 13.
  • the glycerol dehydratase activation factor comprises a polypeptide comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence in SEQ ID NO: 9, 11 or 15.
  • the overexpression of the glycerol dehydratase may increase production of 3-HPA by the genetically engineered L. diolivorans cells compared to wild-type L. diolivorans.
  • the genetically engineered L. diolivorans cells are characterised by an increased production of 3-HPA compared to wild-type L. diolivorans cells.
  • the engineered L. diolivorans may exhibit an increased production of 3-HPA that is at least 10%, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, or more than 5-fold, compared to the wild-type L. diolivorans cells from which they were engineered.
  • the E. coli is engineered to overexpress a 1,3-propanediol oxidoreductase to reduce intracellular accumulation of 3-HPA and increase conversion of 3- HPA to 1,3-PD.
  • the L. diolivorans is also engineered to overexpress a 1,3- propanediol oxidoreductase to increase conversion of 3-HPA to 1,3-PD.
  • the 1,3 -propanediol oxidoreductase may be a bacterial 1,3-propanediol oxidoreductase.
  • the 1,3 -propanediol oxidoreductase may, for example, be derived from Clostridium bulyricum, Lentilactobacillus diolivorans, Citrobacter freundii, Klebsiella pneumoniae, or Clostridium pasleurianum.
  • the 1,3-PD oxidoreductase comprises an amino acid sequence having at least 70% sequence identity (such as about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity) to an amino acid sequence in SEQ ID NO: 17, 19, 21, 23, 25 or 27.
  • the overexpression of the 1,3-PD oxidoreductase may increase production of 1,3-PD by the genetically engineered L. diolivorans or E. coli cells compared to wild-type L. diolivorans or E. coli cells.
  • the engineered L. diolivorans or E. coli is characterised by an increased production of 1,3-PD compared to wild-type L. diolivorans or E. coli cells.
  • coli may exhibit an increased production of 1 ,3-PD that is at least 10%, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, or more than 5-fold, compared to the wild-type L. diolivorans or E. coli cells from which they were engineered.
  • the genetically engineered cells of this disclosure may contain expression constructs encoding the glycerol transporter or glycerol-metabolising enzyme.
  • the expression construct can be stably integrated into the host cell genome or replicated as an episomal vector, such as a plasmid.
  • Suitable vectors will be those which are compatible with the bacteria employed.
  • Expression vectors suitable for use in E. coli are well known in the art.
  • L. diolivorans it may be preferred to use a plasmid originating from wild-type L. diolivorans as an expression vector.
  • a plasmid containing a L. diolivorans origin of replication may be used. Protocols for obtaining and using such vectors are known to those in the art and may be referenced in Sambrook ct al.. Molecular Cloning: A Laboratory Manual — volumes 1, 2, 3 (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989).
  • Codons in the expression construct may be optimised for a particular expression host, as is well known in the art.
  • Genes in the expression construct may be operably linked to one or more regulator ⁇ ' sequences.
  • regulatory sequences include promoters, operators, enhancers, ribosomal binding sites, and sequences that control transcription and translation initiation and termination.
  • the regulator ⁇ ' sequences may be operably linked to the DNA sequence to be expressed.
  • a promoter sequence is said to be operably linked to a coding sequence, if the promotor controls the transcription of the coding sequence.
  • Suitable promoter sequences for use with bacterial host cells herein may include but are not limited to promoters obtained from Lactobacillus sp. , Lactococcus sp. , Staphylococcus sp. , Pediococcus sp., Enterobacteria sp., Streptococcus sp., Oenococcus sp., or E. coli.
  • the promoter is not limited to any particular species provided that they can function in the bacterial host cell.
  • the promoter can be constitutive or regulatable.
  • the regulatable promoters can be, for example, inducible.
  • the promoter sequences can be derived, for example, from the host cell, from another organism, or can be synthetically derived.
  • any desired promoter can be used to regulate the expression of the inserted genes.
  • promoters may be induced by nutrient source, nutrient starvation, growth phase, heat shock, cold shock, oxidative stress, salt stress, environmental stress, metal concentration, specific metabolites or organic compounds, light exposure, etc.
  • the gene(s) may be under the transcriptional control of a constitutive promoter. In this way, a sustained level of transcription and, therefore, enzymatic activity of the corresponding protein can be maintained during the whole period of culture.
  • the constitutive promoter may be endogenous to the host cell. This has the advantage that no recombinant transcription factor has to be present in the host cell.
  • the endogenous promoter is usually well-recognised by the host cell without the need to introduce further genetic modifications.
  • Vectors or expression constructs may be transferred into the host cell by transformation.
  • Preferred methods of transformation for the uptake of the recombinant DNA by bacteria include chemical transformation, electroporation or transformation by protoplastation.
  • Transformants can be obtained by introducing such a vector, c.g. a plasmid, into a host and selecting transformants which express the relevant protein or host cell metabolite with high yields.
  • the polypeptides encoded by the genes can be produced using the recombinant host cell line by culturing a transformant, thus obtained in an appropriate medium, isolating the expressed product or metabolite from the culture, and optionally purifying it by a suitable method.
  • the cell cultures as described herein particularly employ techniques which provide for the production of cell metabolites of interest by biotransformation, such as to obtain the product in the cell culture medium, which is separable from the cellular biomass, herein referred to as “cell culture supernatant”, and may be isolated and optionally purified to obtain the product at a higher degree of purity.
  • Cell culture media provide the nutrients necessary to maintain and grow cells in a controlled, artificial, and in vitro environment. Characteristics and compositions of the cell culture media may vary depending on the particular cellular requirements. Important parameters include osmolality, pH, and nutrient formulations. Feeding of nutrients may be done in a continuous or discontinuous mode according to methods known in the art.
  • Growth and/or production can suitably take place in batch mode, fed-batch mode or continuous mode.
  • a preferred embodiment includes a batch culture to accumulate biomass followed by a fed-batch culture for efficient bioconversion.
  • Substrates or raw materials for bioconversion may be contained in the feed of a fed-batch process, e.g., under substrate-limited conditions, i.e., using limited amounts of the substrate to keep a production cell line under growth-limited conditions and in the production mode.
  • a batch process is a cell culture mode in which all the nutrients necessary for culturing the cells are contained in the initial culture medium, without additional supply of further nutrients during fermentation
  • a feeding phase takes place in which one or more nutrients are supplied to the culture by feeding.
  • the mode of feeding is critical and important, the cell culture and methods described herein are not restricted with regard to a certain mode of cell culture.
  • host cells described herein are cultured in a continuous mode.
  • a continuous fermentation process is characterized by a defined, constant, and continuous rate of feeding of fresh culture medium into a bioreactor, whereby culture broth is at the same time removed from the bioreactor at the same defined, constant, and continuous removal rate. By keeping culture medium, feeding rate and removal rate at the same constant level, the cell culture parameters and conditions in the bioreactor remain constant.
  • L. diolivorans or E. coli culture may occur in two stages: a “growth phase” to accumulate cellular biomass, followed by a “production phase” during which biotransformation occurs.
  • the growth and production phases may be conveniently performed in batch, fed-batch or continuous mode.
  • the medium used for the growth phase can, but need not be, identical to the medium used during the production phase.
  • L. diolivorans may be grown on glucose or xylose for biomass accumulation followed by a feed containing glycerol and optionally vitamin B 12 for biotransformation.
  • the expression of the glycerol transporter and glycerol metabolising enzymes may be initiated during the growth phase.
  • an inducer may be added during the growth phase to induce protein expression prior to the production phase.
  • the inducer may be added only during the production phase.
  • the inducer is added throughout the growth and production phases.
  • the L. diolivorans is cultivated for at least 24 hrs (such as for about 24 hrs, about 30 hrs, about 36 hrs, or longer than 36 hrs) to accumulate biomass prior to sequential culture or co-culture with the E. coli cells.
  • the E. coli is cultivated for at least 8 hrs (such as for about 8 hrs, about 12 hrs, about 16 hrs, about 24 hrs, or longer than 24 hrs) to accumulate biomass prior to sequential culture or co-culture with the L. diolivorans. (a) Sequential culture
  • methods herein comprise sequential culture of the L. diolivorans and E. coli cells.
  • the L. diolivorans cells may be cultivated first in the presence of glycerol to produce a conditioned culture containing 3-HPA and/or 1,3-PD, and the E. coli cells are then cultivated in the conditioned culture to produce 1,3-PD.
  • the Lentilactobacillus diolivorans cells may be cultivated in the presence of glycerol for at least 3 hours to produce 3-HPA and 1,3-PD, such as for about 3 hr, about 4 hr, about 5 hr, or about 6 hr. This is found to provide sufficient time for accumulation of 3-HPA and 1,3-PD for the next stage of bioconversion.
  • Conditioned medium containing 3-HPA and 1,3-PD may be isolated from the L. diolivorans culture for use in the E. coli culture.
  • the medium may be clarified to remove cells, debris and other particulates, and optionally pasteurised or sterilised prior to being used for E. coli culture.
  • the conditioned medium may be supplemented to an E. coli culture (such as an E. coli culture which has undergone expansion to accumulate biomass) to initiate conversion of 3-HPA.
  • the E. coli may be inoculated into the conditioned medium so that both cell growth and 3-HPA bioconversion occurs in the conditioned medium.
  • the conditioned medium is supplemented with additional media components before being used for E. coli culture.
  • the pH of the medium may be adjusted with suitable buffers, and additional carbon and/or nitrogen sources may be added to the medium prior to E. coli culture.
  • the method may comprise cultivating E. coli cells in the L. diolivorans conditioned medium for at least 6 hours, such as for about 6 hr, about 7 hr, about 8 hr, about 9 hr, about 10 hr, about 11 hr, about 12 hr, or longer than 12 hr.
  • Methods herein may comprise co-culture of the genetically engineered L. diolivorans and E. coli cells in the presence of glycerol.
  • Co-culture can be advantageous as it enables immediate consumption of the toxic intermediate 3-HPA by the E. coli cells.
  • the L. diolivorans cells and the E. coli cells may be present at different ratios for the coculture.
  • the E. coli cells comprise at least about 25% of the cells in the co-culture.
  • the E. coli may comprise about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the co-culture.
  • the E. coli cells comprise about 50% of the cells in the co-culture.
  • the amount of E. coli cells present in the co-culture is at least about 25% of the amount of L. diolivorans cells present.
  • the amount of E. coli cells present may be about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 1.2-fold, about 1.4-fold, about 1.6-fold, about 1.8-fold, about 2-fold, about 2.2-fold, about 2.4-fold, about 2.6-fold, about 2.8-fold, or about 3-fold of the amount of L. diolivorans cells present.
  • the amount of E. coli cells present in the co-culture is about the same as (i.e., about 100% of) the amount of L. diolivorans cells present.
  • the L. diolivorans cells may be co-cultured with the E. coli cells for at least 12 hrs for 1,3- PD production.
  • the L. diolivorans cells may be co-cultured with the E. coli cells for about 12 hrs, about 15 hrs, about 18 hrs, about 20 hrs, about 24 hrs, about 28 hrs, about 32 hrs, about 36 hrs, or longer than 36 hrs for 1,3-PD production.
  • the L. diolivorans cells is co-cultured with the E. coli cells for at least 18 hrs.
  • Co-culture may be performed at a temperature between 25°C to 37°C, and may be performed under aerobic or anaerobic conditions. In one embodiment, the co-culture is performed anaerobically at 30°C.
  • the method may comprise isolating 1,3-propanediol from the cell culture of E. coli cells (for sequential culture), or from the co-culture of E. coli and L. diolivorans cells.
  • the medium may be clarified to remove cellular matter, particulates and high molecular weight biomolecules prior to 1 ,3-PD extraction. Clarification may be performed using methods known to the skilled person, such as filtration, microfiltration, ultrafiltration, centrifugation and/or ion exchange chromatography.
  • the medium may be subjected to an evaporation step to reduce the amount of water, for instance, to less than about 25% v/v to facilitate clarification and/or 1,3-PD isolation.
  • the 1 ,3-propanediol can be isolated and purified using techniques known in the art. Solvent extraction, distillation and chromatography are preferred, particularly for preparative isolation.
  • a highly purified product may be produced which is essentially free from contaminating proteins, and preferably has a purity of at least 90%, more preferred at least 95%, or even at least 98%, up to about 100%.
  • the purified products may be obtained by purification of the cell culture supernatant or else from cellular debris.
  • the isolated and purified product can be identified by conventional methods such as high pressure liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), NMR spectroscopy, infrared spectroscopy and Raman spectroscopy.
  • HPLC high pressure liquid chromatography
  • GC gas chromatography
  • MS mass spectrometry
  • NMR spectroscopy infrared spectroscopy
  • Raman spectroscopy Raman spectroscopy
  • kits for biological production of 1,3-propanediol from glycerol comprising Lentilactobacillus diolivorans cells genetically engineered to overexpress a glycerol transporter, and Escherichia coli cells genetically engineered to overexpress a 1,3- propancdiol oxidorcductasc.
  • the L. diolivorans cells are further engineered to overexpress (i) a glycerol dehydratase and a glycerol dehydratase activation factor, and/or (ii) a 1 ,3- propanediol oxidoreductase.
  • the two bacterial strains may be provided separately or in a combined formulation, depending on the intended application.
  • the bacteria cells may be provided in a sterile, stable and storable form.
  • the cells may be provided in freeze-dried (lyophilised) form containing suitable cryoprotectants (e.g., trehalose, sucrose, or skim milk) to preserve viability.
  • suitable cryoprotectants e.g., trehalose, sucrose, or skim milk
  • the cells may also be provided preserved in a cryoprotective solution (e.g., 15- 25% w/v glycerol) and stored at ultra-low temperatures (e.g., -80°C).
  • a cryoprotective solution e.g. 15- 25% w/v glycerol
  • ultra-low temperatures e.g., -80°C
  • Such stocks may be thawed and cultured directly for experimental use.
  • cells may be provided as live cultures stored in semi-solid agar contained
  • the kit may optionally comprise buffers, growth media and/or glycerol feedstocks for bacterial culture. Furthermore, the kit may include instructions for culturing conditions, growth media compositions, and parameters for induction of gene expression, if applicable.
  • E. coli BL21 Bacteria strains and plasmids E. coli BL21 (DE3) was used as a host strain for heterologous expression.
  • E. coli DH5a was used as a host for plasmids propagation.
  • Lenlilaclobacillus diolivorans LMG 19668 was used as a native producer of 1 ,3-PD.
  • pET-28a (+) was used as expression plasmid.
  • the DNA sequence eglpF of glycerol transporter was amplified from E. coli BL21 (DE3).
  • the DNA sequence bglpF of glycerol transporter was amplified from Bacillus subtilis.
  • the DNA sequence pduF of glycerol transporter was amplified from Lentilactobacillus diolivorans DSM 14421.
  • the DNA sequences dhBlB2 of B12-indcpcndcnt glycerol dehydratase and its activation factor was ordered from Twist.
  • the DNA sequence pduQ of 1,3-PD oxidoreductase was amplified from Lentilactobacillus diolivorans DSM 14421. All these genes were cloned into a vector carrying a replication origin of L. diolivorans (repA), under the control of GAP, PGM and P32 promoters of L. diolivorans as well as ampicillin and erythromycin resistance ( Figure 1). In case of the empty vector (EV) control without introducing the glycerol transporter encoding gene.
  • repA replication origin of L. diolivorans
  • Figure 1 In case of the empty vector (EV) control without
  • the cells were finally resuspended in 100 pL electroporation buffer (272 mM sucrose, 7 mM sodium phosphate, 0.5 mM MgCh, pH 7.4). An 80 pL aliquot of cells was mixed with 3 pg DNA and electroporation was carried out at 2.5 KV, 200 , and 25 pF in an electroporation cuvette with 0.4 cm gap width.
  • electroporation the cells were resuspended in 900 pL MRS supplemented with 2% (w/v) glucose, 0.3 M sucrose and 20 mM MgCh. This mixed culture was incubated at 30 °C, 180 rpm for 3 h. After regeneration, spread the culture on a plate supplemented with 5 pg/mL erythromycin to wait for colonies appear.
  • L. diolivorans culture Modified MRS medium containing 5 mg/mL vitamin B12, 5 pg/mL erythromycin, 3% glucose and 1% glycerol was used for L. diolivorans biomass generation.
  • the bacteria were collected and used as whole cell biocatalysts to convert glycerol to 3-HPA and 1,3-PD.
  • the plasmids pET28a-d/ia77, pET28a-d/ia7'2, pET28a-d/M73, pET28a-d/M7'4, pET28a- dhaT5 and pET28a- ⁇ f/ia76 were transferred into competent cells of E. coli BL21(DE3), and the recombinants were named E. coU/ ⁇ . E. colill, E. colU3, E. colUE E. colU5 and E. colitb, respectively.
  • the recombinants were inoculated and cultivated in LB with kanamycin at 37°C.
  • BL21 -d/; «7 was used as whole cells biocatalysts for biotransformation of 3-HPA to 1 ,3-PD ( Figure 2).
  • BL21 -d/iaT were harvested after routinely grown in LB medium and induced by IPTG. Then the cells of BE21-dhaT were incubated in 100 mM potassium phosphate buffer (pH 6.0) at 30 °C for 30 min with 2% TritonX-100 (v/v) for permeabilization and washed twice with 100 mM potassium phosphate buffer (pH 6.0), then were used for the second step biotransformation.
  • L. diolivorans cells were cultured anaerobically at 30°C in MRS broth with 1 g/L glycerol and 30 g/L glucose.
  • Engineered E. coli strains were grown in LB medium (50 mg/L kanamycin) at 37°C until OD600 reached 0.6, induced with 0.2 mM IPTG, and cultured for additional 12 h at 16°C.
  • Cells were harvested by centrifugation (8000 x g, 4°C, 10 min), washed with potassium phosphate buffer (PPB, 0.1 M, pH 7.0).
  • the collected cells of L. diolivorans and E. coli were resuspended in MRS (pH 7) to 10 g/L dry cell weight. Biotransformation was conducted at 30°C under anaerobic condition. Measurement of 3-HPA and 1,3-PD
  • 3-HPA was quantified by the colorimetric method or HPLC. Briefly, 1 mL of the appropriately diluted sample was mixed with 0.75 mL of 10 mM DL-tryptophan solution and 3 mL 37% HC1. 1,3-PD were determined with HPLC analysis (Agilent) with an Aminex HPX-87H column (300 mm x 7.8 mm; Biorad). The column was operated at 55°C and a flow rate of 0.8 mL/min with 5 mM H2SO4 as mobile phase. A refraction index detector (RID) was used for 3-HPA and 1,3-PD detection. The standards of 3- Hydroxypropionaldyhyde, 1,3-propanediol and Sulfuric acid were purchased from Sigma- Aldrich.
  • 3-HPA serves as intermediate in the biosynthesis of 1,3-PD through glycerol metabolism pathway. Therefore, improving the efficiency of 3-HPA biosynthesis is essential for enhancing 1 ,3-PD production.
  • the key stages in 3-HPA production involve the initial uptake of glycerol into the cell followed by its conversion to 3-HPA through glycerol dehydratase. Lactobacillus has been observed to accumulate 3-HPA outside the cell, keeping intracellular levels low and non-toxic, suggesting that export is not the limiting factor. Therefore, limitations associated with glycerol uptake and its subsequent conversion into 3-HPA was primarily investigated.
  • the constitutive GAP promoter (glyceraldehyde- 3 -phosphate dehydrogenase) was utilized to overexpress three glycerol uptake facilitator proteins from different microorganisms: EglpF from E. coli BL21, BglpF from Bacillus subtilis, and pduF native to L. diolivorans DSM 14421, resulting in the generation of LMG19668/eg//?L’, LMG 19668/bglpF, and LMG19668/pduL strains.
  • an empty vector was introduced into L. diolivorans LMG19668, generating LMG19668/EV strain.
  • Module I Stepwise metabolic engineering of L. diolivorans for enhanced 1,3-PD production
  • Lac/1 overexpressing glycerol transporter
  • Lac/2 co-expressing glycerol transporter and B 12- dependent glycerol dehydratase with its activation factor
  • Lac/3 co-expressing glycerol transporter and B12-independent glycerol dehydratase with its activation factor
  • Lac/4 co-expressing glycerol transporter, B 12-independent glycerol dehydratase with its activation factor, and 1,3-PD oxidoreductase
  • Lac/4 achieved the highest 1,3-PD production compared to control strains (wild-type LMG19668 and LMG19668/EV) ( Figure 7a). Further optimization of Lac/4 through response surface methodology improved 1,3-PD production to 18 g/L, though 2.1 g/L 3-HPA accumulation remained ( Figure 7b).
  • EXAMPLE 2 L. diolivorans and E. coli co-culture further enhances 1,3-PD production
  • the dhaT genes (Genebank No.: WP035771780.1, KRL65223.1, AAM54730.1, WP_003441638.1, WP_260222463.1, WP_032937336.1) encoding 1,3-propanediol oxidoreductase (PDOR) from different microorganisms were ordered from T wist and cloned into pET-28a (+), and transformed into E. coli BL21(DE3) for PDOR expression.
  • L. diolivorans is advantageous for bioconversion due to its generally regarded as safe (GRAS) status and high robustness in handling the stresses encountered in industrial bioprocesses.
  • GRAS generally regarded as safe
  • L. diolivorans has limited 1,3-PD production capacity, and glycerol bioconversion in L. diolivorans is often bottlenecked by the accumulation of 3-HPA, mostly due to insufficient expression of PDOR (DhaT).
  • DhaT PDOR
  • glycerol transporter derived from E. coli, B. subtilis and L. diolivorans were overexpressed in L. diolivorans LMG19668 to enhance 3-HPA production.
  • LMG19668/pc?MF as whole-cell biocatalyst
  • module I L. diolivorans generated four strains through stepwise overexpression: Lac/1 (overexpressing glycerol transporter), Lac/2 (overexpressing glycerol transporter and B 12-dependent glycerol dehydratase with its activation factor), Lac/3 (overexpressing glycerol transporter and B12-independent glycerol dehydratase with its activation factor), and Lac/4 (overexpressing glycerol transporter, B 12-independent glycerol dehydratase with its activation factor, and 1,3-PD oxidoreductase).
  • PDOR from different microorganisms were overexpressed in E. coli), generating strains E. coli/1-6. These strains were used as wholecell biocatalyst to increase conversion of 3-HPA to 1,3-PD.
  • the co-cultivation systems were established by combining Module T Lac/4 (5 g/L) with Module TI E. coli strains at various ratios. Among these, co-cultivation system4 (CS4) exhibited optimal performance, particularly at a 1:1 strain ratio, achieving 28.03 g/L 1,3-PD production with minimal 3- HPA accumulation (0.2 g/L).
  • the cells of both engineered L. diolivorans and E. coli can be reused for another round with similar production of 3-HPA and 1.3-PD.

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Abstract

La présente divulgation concerne des procédés de préparation de propane-1,3-diol (1,3-PDO) à partir de glycérine à l'aide de cellules de Lentilactobacillus diolivorans et d'Escherichia coli génétiquement modifiées. La L. diolivorans est modifiée pour surexprimer un transporteur de glycérine, la glycérol déshydratase avec son facteur d'activation et/ou la propane-1,3-diol oxydoréductase, tandis que l'E. coli est modifiée pour surexprimer une propane-1,3-diol oxydoréductase. Dans un procédé de culture, la L. diolivorans est cultivée dans un milieu contenant de la glycérine pour produire du 1,3-PDO et l'intermédiaire 3-hydroxypropionaldéhyde (3-HPA). L'E. coli est ensuite cultivée dans le milieu de culture de L. diolivorans pour convertir le 3-HPA en 1,3-PDO. En variante, les deux types de cellules peuvent être cultivés ensemble en présence de glycérine pour produire du propane-1,3-diol.
PCT/SG2025/050246 2024-04-09 2025-04-09 Procédé de préparation de propane-1,3-diol Pending WO2025216711A1 (fr)

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