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  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the present disclosure relates to a novel architecture of energy redistribution in microbes that can sustain the increased formation of biofuel like isobutanol and key cofactors like NADH/NADPH.
• Genetically modified microorganisms comprising altered genes are disclosed wherein said alteration optionally along with subjecting the genetically modified microorganism to nutrient stress induces redistribution of energy ultimately resulting in maximum production of isobutanol.
• Butanol or butyl alcohol is a primary alcohol with a 4 carbon structure and the molecular formula of C 4 H 9 OH. There are four isomeric structures for butanol.
• the straight chain isomer with the alcohol functional group at the terminal carbon which is also known as n-butanol or 1 -butanol.
• the straight chain isomer with the alcohol at an internal carbon is sec-butanol or 2- butanol.
• the branched isomer with the alcohol at a terminal carbon is isobutanol, and the branched isomer with the alcohol at the internal carbon is tert-butanol; 2-methyl-2-propanol.
• n-Butanol and isobutanol have limited solubility, while the other two isomers are fully miscible with water and hence less suitable as next-generation biofuel.
• isobutanol requires no infrastructure modifications for transport and use because, unlike ethanol, isobutanol is not hygroscopic and is not corrosive to motor engines. It should also be noted that isobutanol can be blended with gasoline at higher ratios (16%) when compared to ethanol (10%), increasing both the green footprint of the blend and the market.
• ABE fermentation method as the name suggests is a method used for the production of Acetone, Butanol (n-butanol) and Ethanol.
• the source used during this fermentation procedure is starch and this fermentation takes place under anaerobic conditions.
• Pasteur produced butanol by biological means for the first time.
• Schardinger produced Acetone in a similar manner.
• Fernbach used starch for the production of n-butanol.
• ABE fermentation began in 1916 with Charles Weizmann's isolation of Clostridium acetobutylicum. These solvents are produced in a ratio of 3-6-1, or 3 parts Acetone, 6 parts Butanol and 1 part Ethanol.
• the bacterium Clostridium acetobutylicum and Clostridium beijerinkii were used to produce these fuels in a moderate industrial scale.
• ABE fermentation however, lost out due to profitability factor when compared to the production of these solvents from petroleum. As such, there are no currently operating ABE plants.
• ABE fermentation was replaced by petroleum chemical plants. Due to different cost structures, ABE Fermentation was viable in South Africa until the early 1980s, with the last plant closing in 1983.
• ABE fermentation process -Production of Acetone, Butanol and Ethanol in the ratio 6:3: 1.
• ABE fermentation process yield only 1.3 gallon Butanol/bushel of corn, where as yeast fermentation produces 2.5 gallon of Ethanol/bushel of corn.
• Butanol is toxic to Clostridium acetobutylicum at the threshold level of 1-2%, thus hindering higher yield.
• the present disclosure relates to a genetically modified micro-organism comprising altered genes, selected from a group comprising ackA, ldhA, adhE and kivD or any combination thereof; a process for obtaining a genetically modified micro-organism comprising altered genes, selected from a group comprising ackA, ldhA, adhE and kivD or any combination thereof, said process comprising altering expression of the genes by: a) knocking out a gene selected from a group comprising ackA, ldhA and adhE, or any combination thereof, or b) engineering of the codon optimized ketoisovalerate decarboxylase (KIVD) sequence set forth as SEQ ID No.
• KIVD ketoisovalerate decarboxylase
• a method for inducing redistribution of energy within a micro-organism comprising act of: (a) altering expression of genes corresponding to biomolecules involved in predetermined biochemical pathways within the micro-organism, or (b) growing the micro- organism under nutrient stress condition, or performing a combination of steps (a) and (b), to induce the redistribution of energy; and a method for producing isobutanol from a genetically modified micro-organism, said method comprising act of: (a) obtaining genetically modified micro-organism comprising altered genes corresponding to biomolecules involved in predetermined biochemical pathways, and (b) optionally, growing the micro-organism obtained in step (a) under nutrient stress condition, to induce the redistribution of energy for production of said isobutanol.
• FIG. 1 depicts minimal pathways required for the production of isobutanol. Note: The enzyme Keto isovalerate decarboxylase (KIVD) is depicted in the figure as kdc.
• KIVD Keto isovalerate decarboxylase
• Figure 2 depicts pyruvate flux distribution to multiple pathways.
• Figure 3 depicts the computational model under simulation showing the simulation relationship between Nitrogen availability versus Biomass production rate when Isobutanol pathway is constrained to divert a given carbon flux.
• Figure 4 depicts the computational model under simulation showing the simulation relationship between the ability for the microbe pathway to sustain a constrained level of carbon diversion into the Iso-butanol pathway when a minimal Nitrogen availability of 10 % of normal requirement is maintained.
• Figure 5 depicts the computational model under simulation showing the simulation relationship between the ability for the microbe pathway with an ackA knockout to sustain a constrained level of carbon diversion into the Iso-butanol pathway when a minimal Nitrogen availability of 10 % of normal requirement is maintained along.
• Figure 6 depicts the computational model under simulation showing the simulation relationship between the ability for the microbe pathway with an adhE knockout to sustain a constrained level of carbon diversion into the Iso-butanol pathway when a minimal Nitrogen availability of 10 % of normal requirement is maintained.
• Figure 7 depicts the computational model under simulation showing the simulation relationship between the ability for the microbe pathway having a combined knockout of adhE, ackA and IdhA to sustain a constrained level of carbon diversion into the Iso-butanol pathway when a minimal Nitrogen availability of 10 % of normal requirement is maintained.
• Figure 8 A depicts the plasmid pUC57.
• Figure 8B depicts the multiple cloning site on the plasmid pUC57.
• Figure 9 depicts the plasmid pUCKl .
• Figure 10 depicts the verification study for double knock out using colony PCR with ackA and adhE primers.
• Lane 1 and 2- ackA without Kan cassette (about 755 bps) with ackA primers Lanes 5 and 7- adhE with Kan cassette with adhE primers (1900 bps)
• Lane 12- NEB 1 kb ladder (sizes in base pairs indicated).
• Figure 11 depicts the verification study for triple knock out using colony PCR with IdhA primers.
• the present disclosure relates to a genetically modified micro-organism comprising altered genes, selected from a group comprising ackA, IdhA, adhE and kivD or any combination thereof.
• the micro-organism is Escherichia coli strain B; and wherein the Escherichia coli strain B is selected from a group comprising Escherichia coli BL21 and Escherichia coli BL21 (DE3).
• the Escherichia coli strain B comprising the altered genes is capable of survival under nitrogen deficient condition.
• the nitrogen deficiency leads to the activation of ilvG gene even under anaerobic condition.
• expression of the gene is altered to facilitate redistribution of energy for optimizing biochemical pathway for production of isobutanol.
• the production of isobutanol is enhanced by at least 2 fold under nitrogen deficient condition.
• the expression of the gene is altered by method selected from a group comprising knock out and engineering of the gene or any combination of method thereof.
• the expression of the gene is altered by knocking out of the gene selected from a group comprising ackA, ldhA and adhE, or any combination thereof; and wherein the expression of the gene is altered by engineering of codon optimized ketoisovalerate decarboxylase (KIVD) sequence set forth as SEQ ID No. 1.
• KIVD ketoisovalerate decarboxylase
• the kivD gene is isolated from bacterium Lactococcus lactis and codon-optimized for Escherichia coli to obtain said sequence.
• the present disclosure further relates to a process for obtaining a genetically modified microorganism comprising altered genes, selected from a group comprising ackA, ldhA, adhE and kivD or any combination thereof, said process comprising altering expression of the genes by: a) knocking out a gene selected from a group comprising ackA, ldhA and adhE, or any combination thereof, or b) engineering of the codon optimized ketoisovalerate decarboxylase (KIVD) sequence set forth as SEQ ID No. 1, or any combination of alterations thereof.
• KIVD ketoisovalerate decarboxylase
• the micro-organism is Escherichia coli strain B; and wherein the Escherichia coli strain B is selected from a group comprising Escherichia coli BL21 and Escherichia coli BL21 (DE3).
• expression of the gene is altered to facilitate redistribution of energy for optimizing the biochemical pathway for production of isobutanol.
• isobutanol is enhanced by at least 2 fold under nitrogen deficient condition.
• the knocking out of the gene is carried out by method comprising acts of: a) growing phage on donor strain for packaging of genome fragment with antibiotic marker to obtain phage lysate, b) infecting recipient strain with the phage lysate to integrate the genome fragment by homologous recombination, and c) identifying the recipient strain through the antibiotic marker to confirm knocking out of gene to obtain single knock out.
• the knocking out of more than one of the genes is carried out by method comprising acts of: a) obtaining single knock out as claimed in claim 14, b) transforming the single knock out with plasmid expressing flippase employed in FLP-FRT system, c) excising gene flanked by the flippase to obtain a knock out of two genes or a double knock out, optionally d) conducting the step b) with the double knock out of step c) to obtain a knock out of three genes or a triple knock out.
• the present disclosure further relates to a method for inducing redistribution of energy within a micro-organism, said method comprising act of: (a) altering expression of genes corresponding to biomolecules involved in predetermined biochemical pathways within the micro-organism, or (b) growing the micro-organism under nutrient stress condition, or performing a combination of steps (a) and (b), to induce the redistribution of energy.
• the alteration of expression of genes comprises acts of: a) identifying biochemical pathway responsible for distribution of energy within a microorganism, b) identifying biomolecule participating in said distribution of energy and the corresponding gene involved in the pathway of step a), c) altering expression of said genes to modulate the participation of said biomolecules for inducing said redistribution of energy.
• the nutrient stress is caused by deficiency of nitrogen in conditions employed for the growth of micro-organism.
• the micro-organism is Escherichia coli strain B; and wherein the Escherichia coli strain B is selected from a group comprising Escherichia coli BL21 and Escherichia coli BL21 (DE3).
• the identification of the biochemical pathway and said biomolecules is carried out by conventional methods; and wherein the biomolecule is selected from a group comprising NADH, NAD, NADPH, NADP, ATP, ADP, GTP, GDP, FADH, FAD, Pyruvate, Ubiquinone and Acetyl CoA or any combination thereof.
• the gene involved in the biochemical pathway responsible for distribution of energy with the microorganism is selected from a group comprising ackA, ldhA, adhE and kivD or any combination thereof.
• the expression of the gene is altered by method selected from a group comprising knock out, overexpression and engineering of the gene or any combination of method thereof.
• the expression of the gene is altered by knocking out of the gene selected from a group comprising ackA, ldhA and adhE or any combination thereof; and wherein the expression of the gene is altered by engineering of codon optimized ketoisovalerate decarboxylase (KIVD) sequence set forth as SEQ ID No. 1.
• KIVD ketoisovalerate decarboxylase
• the redistribution of energy results in sustainable biomass levels for production of isobutanol.
• the present disclosure further relates to a method for producing isobutanol from a genetically modified micro-organism, said method comprising act of: (a) obtaining genetically modified micro-organism comprising altered genes corresponding to biomolecules involved in predetermined biochemical pathways, and (b) optionally, growing the micro-organism obtained in step (a) under nutrient stress condition, to induce the redistribution of energy for production of said isobutanol.
• the micro-organism is Escherichia Coli strain B; and wherein the Escherichia coli strain B is selected from a group comprising Escherichia coli BL21 and Escherichia coli BL21 (DE3).
• the genetically modified micro-organism comprise altered genes, selected from a group comprising ackA, ldhA, adhE and kivD or any combination thereof.
• the redistribution of energy within the microorganism is induced by method as described above.
• the nutrient stress is caused by deficiency of nitrogen in conditions employed for the growth of micro-organism.
• the method enhances the production of the isobutanol at least by 2 fold when compared to the production of the isobutanol by wild type microorganism without said redistribution of energy.
• Any heterologous expression involving the production of a foreign molecule in a microorganism like E.coli requires a suitable nutrient source and redistribution of energy for optimal production.
• high energy is generally associated with Pyruvate, Acetyl Coenzyme A, ATP and NADH and NADPH.
• the present disclosure focuses on optimizing the yield of isobutanol by redistributing the flux of these molecules by altered nutrient source, specific knockouts, expressions of desired homologous/heterologous genes or by any combination of said aspects.
• BCAA branched chain amino acid
• Figure 1 illustrates the minimal pathways required for the production of isobutanol. It is well accepted that in all living cells including microbes, the pathway from Glucose to ketoisovalerate is generally present. The first phase of the pathway which is from glucose to pyruvate, is a part of the Glycolytic pathway, while the second phase, which is from pyruvate to ketoisovalerate is a part of the amino acid valine synthesis pathway. The two enzymes viz.
• keto iso valerate decarboxylase KIVD
• ADH specific alchohol dehydrogenase whose substrate is isobutanal
• ketoisovalerate decarboxylase KIVD
• ADH specific alchohol dehydrogenase whose substrate is isobutanal
• YqhD, AdhP, FucO, EutG, YaiY, BetA, EutE, YjbB genes of the alcohol dehydrogenase family which can convert the penultimate metabolite isobutanal to isobutanol with different catalytic efficiency and cofactor specificity.
• the biomass is generated by standard media (such as M9+ Glucose, Luria Bertani, etc.) and thereafter, the culture is shifted to a specialized media which has either zero or limited amount of nitrogen. Due to this shift, there is a large diversion of flux towards the production of isobutanol since all other pathways require the element Nitrogen. This aspect of shift of media wherein all other constituents except Nitrogen are at optimum concentration is termed as "Nitrogen Swap" (N-Swap).
• nitrogen is an essential molecule for cellular growth and is a part of proteins and DNA. Thus, under a condition of nitrogen swap, the growth of the cell is halted. However, since other nutrients like carbon, hydrogen, oxygen etc. are present in the media, the metabolite flux will only proceed through Nitrogen independent pathway.
• Table 1 List of metabolites that are formed from glucose, i.e. glucose-6-phosphate to isobutanol
• N-swap as described in the present disclosure is that under limiting or no nitrogen conditions, the NO formation is effectively stunted which helps in keeping the flux active through the BCAA pathway.
• This complex has been shown to be activated under aerobic condition without the formation of new enzyme.
• the present disclosure describes an in-silico formulation to identify the gene deletions/alterations that maximize the production of isobutanol without compromising the biomass formation.
• a self-sustaining balanced system is achieved through an in-silico simulation of a computational model of the microbial cell.
• the engineering/ re-construction of the strain (gene deletions and over-expressions in specific sections of the microbial pathways in the cell) is estimated so that possible strains that have the simultaneous capability of growth and yield of the product viz. isobutanol can be identified and subsequently constructed.
• Another illustrative embodiment of the present disclosure relates to a genetically modified micro-organism comprising combination of biochemical pathways for redistribution of energy for the optimum production of isobutanol.
• the embodiment relates to a method for inducing redistribution of energy within a micro-organism, said method comprising steps of- a) identifying biochemical pathway(s) to be optimized within said organism, b) identifying native genes involved in the pathway(s) of step (a) along with introducing heterologous genes for optimizing the pathway(s) within the organism, and
• step (b) altering the expression of genes of step (b) for inducing redistribution of energy.
• the present disclosure relates to a method for producing isobutanol from a genetically modified micro-organism, said method comprising steps of- a) identifying native genes involved in biochemical pathway(s) within the organism, b) introducing heterologous genes for biochemical pathway(s) of the metabolite in the micro-organism and
• the in-silico simulation of whole cell functioning and its response to internal and external perturbations at the molecular and kinetic detail is carried out.
• Such an in-silico model is a computational model of the E.coli. With the genome of a number of organisms sequenced and nearly entire metabolic pathway constructed in chemical detail, what remains is the dovetailing of the kinetic to the static pathway platform.
• the first simulation of the bacterium E.coli computational model is successfully demonstrated with the platform of the present disclosure. In this platform the control of the enzymatic and pathway functioning is simulated by interconnecting the behaviour of each enzyme in the pathway translating as an ability to sustain a rate of reaction flow with the necessary regulation parameters that provide the crosstalk between these enzymes as feedback and feed forward mechanisms, controlling growth from a given carbon source.
• This computational mathematical framework built by using intercellular enzyme concentration and other control parameters responds in a similar fashion to perturbations the way the natural system in question would.
• This type of modelling has the ability to solve systems comprising of unlimited number (in thousands) of simultaneous control pathways interconnected in a complex way and able to maintain stoichiometry and provide a test platform for a given carbon source of a given mole quantity.
• the predictive power of this platform in E.coli is experimentally validated. Enzymes in a number of pathways including TCA, Glycolytic, Glyoxylate bypass, Branched chain amino acid synthesis, CoA biosynthesis, Nucleotide Biosynthesis and Nicotinamide Biosynthesis pathway are evaluated.
• Enzymes in pathways that are either vulnerable or relatively immune to inhibition of a specific type are delineated and experimentally corroborated.
• the disclosure herein show an example set of computations in usage of this computational model for isobutanol production from Glucose and the carbon source and the predicted changes that need to be done in re-engineering this organism to enable higher isobutanol yields and limits of mole to mole conversion of glucose to isobutanol.
• the present disclosure is a continuum of the above wherein the concept of nutrient swap is tested out to identify which constructs including Knockouts and overexpressions yield the best production of isobutanol.
• the disclosure herein show an example set of computations in usage of this computational model for isobutanol production from Glucose and the carbon source and the predicted changes that need to be done in re-engineering the organism to enable higher isobutanol yields and limits of mole to mole conversion of glucose to isobutanol.
• a computational systemic model of the microorganism model is used to study the effects of nitrogen depletion referred as the "Nitrogen Swap" and the diversion of the pyruvate flux into the targeted Isobutanol pathway.
• the computational model enables detection of limits of a possible computational solution to the system for producing Isobutanol against constrained conditions for growth.
• the solution set on this computational model for different constrained growth rates provide quantitative estimates of the nitrogen requirements to sustain the constrained growth.
• Figure 3 shows the computational model under simulation showing the simulation relationship between Nitrogen availability Vs. Biomass production rate when Isobutanol pathway is constrained to divert a given carbon flux. '301 ' shows the reference biomass production with respect to Nitrogen availability as shown when there is no diversion of carbon flux through the Isobutanol pathway for computational analysis. This is treated as the control reference. Nitrogen availability is normalized to the maximum level needed with unit Glucose flow through the simulation model. The carbon source in this simulation is exclusively glucose. The simulation analysis aims at proving the biomass growth trends with various levels of Nitrogen available in the growth medium.
• a second variation of constraining the model to force a percentage of the carbon flux through the isobutanol pathway is also made in the simulation to simulate biomass growth for these constraints for all selections of Nitrogen availability. Accordingly, '302' shows that with a 15 % re-routing carbon flux from Glucose through the Isobutanol pathway. Likewise, '303', '304' and '305' show the variation of Biomass production with respect to availability of nitrogen, 30%,45% and 60% respectively. The simulation results show that, for the computational model growth directly depends on availability of Nitrogen and increasing carbon diversion into the isobutanol pathways cuts back growth.
• Figure 3 depicting the computational analysis provides a relationship between Nitrogen availability, Biomass production rate and the effect on the diversion of additional carbon flux available towards Isobutanol pathway.
• “Figure 4" shows the computational model under simulation depicting the simulation relationship between the ability of the microbe pathway to sustain a constrained level of carbon diversion into the Iso-butanol pathway when a minimal Nitrogen availability of 10 % of normal requirement is maintained.
• the computational analysis further evaluates, with respect to the specific constrained minimum carbon flux made to flow through the Isobutanol pathway, the various other pathways like Acetate, Lactate, Etanol and carbon-dioxide etc. into which the glucose carbon source is also utilized as part of the carbon metabolism of the microbe.
• An assessment is made on this sum total of diversion, other than the iso-butanol pathway.
• This pooled diversion, in summation, is represented also in arbitrary units by 401 .
• “Degree Of Freedom” (DOF) is then defined as the quantitative estimate of a part of this pooled diversion that can be re-routed through the iso-butanol pathway by specific gene deletions to shut down respective parts of the pathways that constitute the summation previously estimated.
• 402 shows this DOF which is available to re-route additional carbon flux through the iso-butanol pathway. From this figure-4, it is estimated that for a constraint of diverting over 25 % carbon flux through the iso-butanol pathway, the DOF rapidly drops to zero from a sustainable limit of about 20 % through the isobutanol pathway. Over this range of constrained iso-butanol use of carbon from glucose feed, 402 remains reasonably constant signifying that the model predicts a stable level of producing other metabolites that represent carbon utilization including the major metabolites like Acetate , Lactate , Ethanol and Carbon-dioxide etc. Thus, in summary, Figure 4 indicates that microbe sustains when up to 20 % of carbon flux available is re-routed for 'Isobutanol production'.
• Figure 5" shows the computational model under simulation showing the simulation relationship between the ability for the microbe pathway to sustain a constrained level of carbon diversion into the Iso-butanol pathway when a minimal Nitrogen availability of 10 % of normal requirement is maintained and an ackA knockout is carried out.
• '502' shows the "Degree Of Freedom” (DOF) that is available to re-route additional carbon flux through the iso-butanol pathway with this knockout.
• DOF Degree Of Freedom
• ' Figure 5' depicts that with a combination of low Nitrogen availability of 10 % and ackA knockout, acetate production is substantially reduced and up to 45% additional carbon flux available can be diverted for isobutanol production.
• Figure 6 shows the computational model under simulation showing the simulation relationship between the ability for the microbe pathway to sustain a constrained level of carbon diversion into the Iso-butanol pathway when a minimal Nitrogen availability of 10 % of normal requirement is maintained and an adhE knockout is carried out.
• '602' shows the "Degree Of Freedom” (DOF) which is available to re-route additional carbon flux through the iso-butanol pathway with this knockout.
• DOE Degree Of Freedom
• Figure 7 shows the computational model under simulation showing the simulation relationship between the ability for the microbe pathway to sustain a constrained level of carbon diversion into the Iso-butanol pathway when a minimal Nitrogen availability of 10 % of normal requirement is maintained and a combined knockout of adhE, ackA and ldhA is carried out.
• '702' shows the "Degree Of Freedom” (DOF) that is available to re-route additional carbon flux through the iso-butanol pathway with this knockout set.
• DOF Degree Of Freedom
• ' Figure 7' depicts that with a combination of low Nitrogen availability of 10 % and (adhE+ ackA+ ldhA gene knockouts), up to 60% additional carbon flux available can be diverted for isobutanol production. Further, it is established that a combination of gene knock-outs results in a synergistic affect (i.e. 60%> carbon diversion) when compared to individual knock-outs (45 and 50%> respectively).
• the in-silco computational simulation results as described above establishes that the yield of isobutanol can be maximized by redistributing the carbon flux which can be achieved by various factors such as altered nutrient source (Nitrogen availability), specific gene knockouts, expressions of desired homologous/heterologous genes or by any combination of said factors.
• the present disclosure utilizes a codon optimized kivD gene sequence represented by SEQ ID NO. 1 and cloning the said sequence into plasmid puc57, to obtain a cloned plasmid named pucKl .
• the in-vitro model comprising E.coli single knock outs are generated by way of PI transduction system using PI lysate and FLP-FRT system.
• double and triple knock outs are also generated using the PI transduction protocol.
• E. coli BL21 has not been sequenced till date, a closely related variant, E.coli BL21(DE3) has been.
• the two strains are represented as follows:
• E. coli BL21 (DE3) is an E. coli B strain with DE3, i.e., a ⁇ prophage carrying the T7 RNA polymerase gene and laclq.
• E. coli BL21 (DE3) transformed plasmids containing T7 promoter driven expression are repressed until IPTG induction of T7 RNA polymerase from a lac promoter.
• T7 RNA polymerase has been inserted into E.coli BL21 to construct the strain BL21 (DE3).
• the T7 RNA polymerase gene sequence is provided as SEQ ID No. 7.
• the aspects of the present disclosure do not require a T7 promoter driven expression, or an IPTG inducible system, the T7 RNA polymerase gene sequence is not required by the genetically modified E. coli obtained in the present disclosure. However, even when such T7 RNA polymerase gene sequence is not required for the aspects of the instant disclosure, it is also noted that the presence of such T7 RNA polymerase gene sequence will not adversely affect the aspects of the instant disclosure in any manner.
• the genetically modified organism obtained in the present disclosure is a genetically modified E. coli BL21.
• the said genetically modified organism is BL21 (DE3) minus the T7 RNA polymerase gene sequence.
• the knocking out of genes within the purview of the instant disclosure requires deletion of the entire gene sequence, from start codon to the respective stop codon, and re-joining the remaining sequence in order to obtain a knocked-out sequence.
• the native form of the microorganism strain is known [as mentioned above]
• the sequences of the genes to be knocked out is also provided, a person skilled in the art will have no problem or no undue experimental burden in carrying out the procedure of the instant disclosure and to arrive at the final genetically modified organism of the instant disclosure.
• the overexpression and engineering of the genes within the purview of this disclosure requires overexpression and/or inserting specific genes within a native form of the microorganism strain.
• specific genes are provided in the instant disclosure and hence, a person skilled in the art will have no problem or no undue experimental burden in carrying out the procedure of the instant disclosure and to arrive at the final genetically modified organism of the instant disclosure.
• Knock-outs selected from a group of ackA, adhE, ldhA or any combination of knockouts are generated using PI transduction and homologous recombination by linear DNA, after which clones are picked and the specific gene deletions confirmed by PCR.
• Competent cells carrying the various knock-outs are prepared.
• Plasmid containing codon optimized KIVD under inducible lac promoter is transformed into the desired knock-out and plated on ampicillin plates (50-100 ug/ml) and incubated.
• the cultures are incubated for about 3 - 4 hours at about 37 degrees and about 200 rpm.
• the O.Ds of the cells are monitored after which the cultures are divided (depending on various conditions and time points of swap) followed by centrifugation at about 4000 g, and at 20 degrees for about 7-10 minutes.
• the pellets obtained post centrifugation are re-suspended in nitrogen deficient media [ M9+(0%,1%,3%,10%)N +3.6%Glucose + Amp 100].
• All cultures are transferred to 250 ml screw capped conical flasks, they are shut tight and their mouths are parafilmed to maintain an anerobic environment.
• the KIVD (keto isovalerate decarboxylase) is codon optimized from L.lactis. It is known that there is a bias for usage of the degenerate codon among each organism. The codon for the highly expressed genes are different from the moderate and low/lesser expressed genes. The concentration of tRNA in the cell is directly proportional to the codon usage ( Ikemura, T. (1981) J. Mol Biol. 146,1-21; Dong, H., Nilsson, L. and Kurland,C. G. (1996) J. Mol Biol 260,649-663; Kane, J.F. (1995) Curr.Opin.
• CODON OPTIMISED kivD [SEQ ID NO: 1] THE GENE STARTS FROM THE SECOND LINE
• the entire sequence is cloned in the plasmid pUC57.
• the clone is named pUCKl .
• the plasmid pUC57 is 2710 bp in length and is a derivative of pUC19.
• pUC57 MCS multiple cloning site
• This vector is designed for cloning and generating ExoIII deletions. The exact position of genetic elements is shown on the map- Figure 8 (termination codons included).
• DNA replication initiates at position 890 (+/- 1) and proceeds in indicated direction.
• the bla gene nucleotides 2510-2442 (compl. strand) code for a single peptide.
• pUC 57 Sequence wild type: [SEQ ID NO: 2]
• the promoter sequence (-10) TATAAT and -35 (TTACGA) , the start codon (ATG) for KIVD and its stop codon (TAA) are marked in bold.
• PI transduction homologous recombination To move portions of E.coli genome from one variant to another and to generate knock outs, PI transduction homologous recombination is employed.
• phage is first grown on a donor strain during which host genome fragments of about lOOkb along with selectable antibiotic markers when present are packaged in them resulting as a phage lysate.
• This lysate is used to infect recipient strains that would incorporate into their chromosomes foreign bacterial DNA by means of homologous recombination.
• the selection markers aid the tracking of transduced fragments of DNA.
• Recipient strain is grown overnight. About 5ml culture is centrifuged at about 2000-3000 x g and resuspended in about 2.5ml of PI solution (lOmM CaC12 + 5mM MgS04). Thereafter, about 100 micro litres of these cells is mixed in PI with about 1, 10, and 100 micro litres of phage lysate in different tubes and a control is included without phage lysate. This mixture is further incubated for about 20 minutes at about 37°C. To the obtained mixture, about 200 micro litres of 1M Na-citrate and about 1ml LB is added and incubated for about 1 hour at about 37°C.
• ackA which prevents formation of acetyl phosphate and acetate from pyruvate
• adhE codes for alcohol dehydrogenase which would act on acetyl CoA and prevents further action on acetaldehyde. This in-turn increases the production of ethanol.
• the single gene knockout strains that are part of the Keio collection (Baba et al 2006. paper and Datsenko & Wanner , 2000) are obtained from E.coli Genetic Resources at Yale CGSC. To make double gene knockouts, it is essential to remove the Kanamycin marker cassette. A simple strategy involving the use of FLP-FRT system is engineered into the knockouts of the Keio collection.
• pCP20 a temperature sensitive plasmid that expresses a flippase is transformed into a knockout and incubated overnight at 30°C with ampicillin (30ug/ml).When colonies from this are grown under permissible temperatures, the flippase recognizes the FRT sites that flank the kanamycin cassette and excises it out, leaving a marker less knockout. The plasmid is eliminated by selecting the knockouts on antibiotic free conditions at 43°C.
• Methodology for obtaining triple knockout- ackA knockout is first generated as aforementioned. Thereafter, pCP20 DNA is transformed into BL21 ackA knockout and plated overnight in the presence of ampicillin at about 30°C. The following day a few colonies are grown in different snap-cap tubes in the presence of antibiotic ampicillin at about 30°C till they reach an OD of about 0.6. The colonies are then transferred to about 37°C for about 2 hours. These colonies are then diluted into fresh LB and left at about 43°C without ampicillin for about 4 hours. The colonies so diluted are replica plated on LB, Kan30 (30ug/ml) and Amp 100 (lOOug/ml) plates. If the colonies grow only on LB plates, then it means we are able to successfully flip out Kan cassette and can proceed for second knockout after verifying with colony PCR.
• the yield as provided in the above Table 2 is estimated as the amount of isobutanol produced in ppm per ml. 10 %, 3 % and 0 % N-swap indicates the amount of nitrogen in the media compared to the normal concentration (100 %) which is about 5gm/L of NH 4 CI.
• Table 2 clearly establishes the increased isobutanol production in single knock-outs (BL21ack), double knock-outs (BL21ack/adhE) and triple knock-outs (BL21ack/adhE/ldhA) when compared to the control (i.e. E.coli BL21).
• the present disclosure is able to successfully overcome the various deficiencies of prior art and provide for genetically improved microorganisms possessing increased metabolite production characteristics (such as isobutanol), especially when grown under nutrient stress conditions.
• Such genetically modified microorganisms and the methods of present disclosure can be successfully employed to produce various metabolites of interest, especially isobutanol for its application as biofuel.

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