CLONING AND EXPRESSION OF THE GENES ENCODING KEY CLOSTRIDIAL CATALYZING MECHANISMS FOR SYNGAS TO ETHANOL PRODUCTION AND FUNCTIONAL CHARACTERIZATION THEREOF

RELATED U.S. APPLICATION DATA

This application claims the benefit of and priority to United States Patent Application 12/336,238 filed Dec. 16, 2008 as a continuation-in-part application. The entirety of that application is incorporated by reference herein.

FIELD OF THE INVENTION

[0001] This invention relates to the cloning and expression of novel genetic sequences of microorganisms used in the biological conversion of CO, H ₂ , and mixtures comprising CO and/or H ₂ to biofuel products.

BACKGROUND

[0002] Synthetic gas (syngas) is a mixture of carbon monoxide (CO) gas, carbon dioxide (C0 ₂ ) gas, and hydrogen (H ₂ ) gas, and other volatile gases such as CH ₄ , N ₂ , NH ₃ , H ₂ S and other trace gases. Syngas is produced by gasification of various organic materials including biomass, organic waste, coal, petroleum, plastics, or other carbon containing materials, or reformed natural gas.

[0003] Acetogenic Clostridia microorganisms grown in an atmosphere containing syngas are capable of absorbing the syngas components CO, C0 ₂ , and H ₂ and producing aliphatic C ₂ -C ₆ alcohols and aliphatic C ₂ -C ₆ organic acids. These syngas components activate Wood-Ljungdahl metabolic pathway 100, shown in FIG. 1, which leads to the formation of acetyl coenzyme A 102, a key intermediate in the pathway. The enzymes activating Wood-Ljundahl pathway 100 are carbon monoxide dehydrogenase (CODH) 104 and hydrogenase (H ₂ ase) 106. These enzymes capture the electrons from the CO and H ₂ in the syngas and transfer them to ferredoxin 108, an iron-sulfur (FeS) electron carrier protein. Ferredoxin 108 is the main electron carrier in Wood-Ljungdahl pathway 100 in acetogenic Clostridia, primarily because the redox potential during syngas fermentation is very low (usually between -400 and -500 mV). Upon electron transfer, ferredoxin 108 changes its electronic state from Fe ³⁺ to Fe ²⁺ . Ferredoxin-bound electrons are then transferred to cofactors NAD ⁺ 110 and NADP ⁺ 112 through the activity of ferredoxin oxidoreductases 114 (FORs). The reduced nucleotide cofactors (NAD ⁺ and NADP ⁺ ) are used for the generation of intermediate compounds in Wood-Ljungdahl pathway 100 leading to acetyl-CoA 102 formation.

[0004] Acetyl-CoA 102 formation through Wood-Ljungdahl pathway 100 is shown in greater detail in FIG. 2. Either C0 ₂ 202 or CO 208 provide substrates for the pathway. The carbon from C0 ₂ 202 is reduced to a methyl group through successive reductions first to formate, by formate dehydrogenase (FDH) enzyme 204, and then is further reduced to methyl tetrahydrofolate intermediate 206. The carbon from CO 208 is reduced to carbonyl group 210 by carbon monoxide dehydrogenase (CODH) 104 through a second branch of the pathway. The two carbon moieties are then condensed to acetyl- CoA 102 through the action of acetyl-CoA synthase (ACS) 212, which is part of a carbon monoxide dehydrogenase (CODH/ ACS) complex. Acetyl-CoA 102 is the central metabolite in the production of C ₂ -C6 alcohols and acids in acetogenic Clostridia. [0005] Ethanol production from Acetyl CoA 102 is achieved via one of two possible paths. Aldehyde dehydrogenase facilitates the production of acetaldehyde, which is then reduced to ethanol by the action of primary alcohol dehydrogenases. In the alternative, in homoacetogenic microorganisms, an NADPH-dependent acetyl CoA reductase ("AR") facilitates the production of ethanol directly from acetyl CoA.

[0006] Wood-Ljungdahl pathway 100 is neutral with respect to ATP production when acetate 214 is produced (FIG. 2). When ethanol 216 is produced, one ATP is consumed in a step involving the reduction of methylene tetrahydrafolate to methyl tetrahydrofolate 206 by a reductase, and the process is therefore net negative by one ATP. The pathway is balanced when acetyl-P0 ₄ 218 is converted to acetate 214.

[0007] Acetogenic Clostridia organisms generate cellular energy by ion gradient- driven phosphorylation. When grown in a CO atmosphere, a transmembrane electrical potential is generated and used to synthesize ATP from ADP. Enzymes mediating the process include hydrogenase, NADH dehydrogenases, carbon monoxide dehydrogenase, and methylene tetrahydrofolate reductase. Membrane carriers that have been shown to be likely involved in the ATP generation steps include quinone, menaquinone, and cytochromes.

[0008] The acetogenic Clostridia produce a mixture of C ₂ -C ₆ alcohols and acids, such as ethanol, n-butanol, hexanol, acetic acid, and butyric acid, that are of commercial interest through Wood-Ljungdahl pathway 100. For example, acetate and ethanol are produced by C. ragsdalei in variable proportions depending in part on fermentation conditions. However, the cost of producing the desired product, an alcohol such as ethanol, for example, can be lowered significantly if the production is maximized by reducing or eliminating production of the corresponding acid, in this example acetate. It is therefore desirable to metabolically engineer acetogenic Clostridia for improved production of selected C ₂ -C6 alcohols or acids through Wood-Ljungdahl pathway 100 by modulating enzymatic activities of key enzymes in the pathway.

SUMMARY OF THE INVENTION

[0009] One aspect of the present invention provides novel sequences for three key operons which code for enzymes that catalyze the syngas to ethanol metabolic process: one coding for a carbon monoxide dehydrogenase, a membrane-associated electron transfer protein, a ferredoxin oxidoreductase, and a promoter; a second operon coding for an acetate kinase, phosphotransacetylase, and a promoter, and a third operon coding for an acetyl Co A reductase and a promoter.

[0010] Another aspect of the invention provides an isolated vector or transformant containing the polynucleotide sequence coding for the operons described above.

[0011] Another aspect of the invention provides a method of producing ethanol comprising: isolating and purifying anaerobic, ethanologenic microorganisms carrying the polynucleotides coding for an operon comprising carbon monoxide dehydrogenase, a membrane-associated electron transfer protein, a ferredoxin oxidoreductase, and a promoter; an operon coding for an acetate kinase, phosphotransacetylase, and a promoter, or an operon coding for an acetyl CoA reductase and a promoter; fermenting syngas with said microorganisms in a fermentation bioreactor; providing sufficient growth conditions for cellular production of NADPH, including but not limited to sufficient zinc, to facilitate ethanol production from acetyl CoA. [0012] Another aspect of the invention provides a method of producing ethanol by isolating and purifying anaerobic, ethanologenic microorganisms carrying the

polynucleotide coding for acetyl coenzyme A reductase; fermenting syngas with said microorganisms in a fermentation bioreactor; and providing sufficient growth conditions for cellular production of NADPH, including but not limited to sufficient zinc, to facilitate ethanol production from acetyl CoA.

[0013] Yet another aspect of the present invention provides a method of increasing ethanologenesis or the ethanol to acetate production ratio in a microorganism containing the nucleotide sequence(s) coding for one of more of the operons described above, said method comprising: modifying, duplicating, or downregulating a promoter region of said nucleotide sequence to increase the activity of the Acetyl Coenzyme A reductase of Claim 16, or to cause overexpression or underexpression of the nucleotide sequence.

[0014] The present invention is illustrated by the accompanying figures portraying various embodiments and the detailed description given below. The figures should not be taken to limit the invention to the specific embodiments, but are for explanation and understanding. The detailed description and figures are merely illustrative of the invention rather than limiting, the scope of the invention being defined by the appended claims and equivalents thereof. The drawings are not to scale. The foregoing aspects and other attendant advantages of the present invention will become more readily appreciated by the detailed description taken in conjunction with the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is a diagram illustrating the electron flow pathway during syngas fermentation in acetogenic Clostridia including some of the key enzymes involved in the process;

[0016] FIG. 2 is a diagram illustrating the Wood-Ljungdahl (Ci) pathway for acetyl- CoA production and the enzymatic conversion of acetyl-CoA to acetate and ethanol;

[0017] FIG. 3 is a diagram illustrating a genetic map containing the location of one of the carbon monoxide dehydrogenase (CODH) operons which includes cooS, cooF and a ferredoxin oxidoreductase (FOR), in accordance with the invention;

[0018] FIG. 4 is a diagram illustrating the Wood-Ljungdahl pathway for ethanol synthesis and showing a strategy for specifically attenuating or eliminating acetate production in acetogenic Clostridia by knocking out the genes encoding acetate kinase (ack) and phosphotransacetylase (pta) or by modulating acetate production by mutating or replacing the promoter driving phosphotransacetylase and acetate kinase gene expression, in accordance with the invention;

[0019] FIG. 5 is a diagram of the Wood-Ljungdahl pathway for ethanol synthesis, and shows a strategy for specifically increasing ethanol production in C. ragsdahlei by overexpression of an acetyl CoA reductase in a host knocked out for acetate kinase or phosphotransacetylase activity, in accordance with the invention;

[0020] FIG. 6 s a diagram demonstrating the results of the functional assay wherein the spectrophotometric activity of the gene encoding the acetyl CoA reductase was measured after being ligated into the EcoRI site to form pCOS54. DETAILED DESCRIPTION

[0021] The present invention is directed to novel genetic sequences coding for acetogenic Clostridia micro-organisms that produce ethanol and acids from syngas comprising CO, C02, H2, or mixtures thereof.

[0022] Several species of acetogenic Clostridia that produce C ₂ -C6 alcohols and acids via the Wood-Ljungdahl pathway have been characterized: C. ragsdahlei, C. ljungdahlii, C. carboxydivorans, and C. autoethanogenum. The genomes of three of these microorganisms were sequenced in order to locate and modify the portions of the genome that code for the enzymes of interest.

[0023] The genes that code for enzymes in the Wood-Ljungdahl metabolic pathway and ethanol synthesis identified in the C. ragsdahlei genome are presented in Table 1. The first column identifies the pathway associated with each gene. The gene identification numbers indicated in the second column correspond to the numbers representing the enzymes involved in the metabolic reactions in the Wood-Ljungdahl pathway shown in FIG. 1 and FIG. 2.

[0024] Sequence analysis of the C. ljungdahlii genome was conducted. Genes coding for enzymes in the Wood-Ljungdahl pathway, ethanol and acetate production, and electron transfer have been identified and located within the genome. The results are presented in Table 2.

[0025] Similarly, the genome of C. carboxydivorans was sequenced, and genes coding for the enzymes in the Wood-Ljungdahl pathway and ethanol and acetate synthesis were identified and located. The results are presented in Table 3.

[0026] Genes that code for enzymes in the electron transfer pathway include carbon monoxide dehydrogenase, Enzyme Commission number (EC 1.2.2.4). Five separate open reading frame (ORF) sequences were identified in C. ragsdalei and C. ljungdahlii, and six were identified in the C. carboxidivorans genome for the carbon monoxide dehydrogenase enzyme.

[0027] FIG. 3 is a diagram of carbon-monoxide dehydrogenase operon 300. The gene order within operon 300 is highly conserved in all three species of acetogenic Clostridia, and comprises the genes coding for the carbon monoxide dehydrogenase (cooS) (Gene ID 4, Tables 1, 2, and 3), followed by the membrane-associated electron transfer FeS protein (cooF) (Gene ID 55, Table 1; Gene ID 51, Table 2; Gene ID 67, Table 3), in turn, followed by ferredoxin oxidoreductase (FOR).

[0028] A comparison was conducted of the genetic sequence found in the operon of FIG. 3 across the three species of acetogenic Clostridia. The cooS gene had 98% identity between c. ragsdalei and c. ljungdahlii, 84% identity between c. carboxydivorans and c. ragsdahlii, and 85% identity between c. carboxydivorans and c. ljungdahlii. The cooF gene had 98%> identity between c. ragsdalei and c. ljungdahlii, 80%> identity between c. carboxydivorans and c. ragsdalei, and 81% identity between c. carboxydivorans and c. ljungdahlii. The FOR gene had 97% identity between c. ragsdalei and c. ljungdahlii, 77% identity between c. carboxydivorans and c. ragsdalei, and 77% identity between c.

carboxydivorans and c. ljungdahlii.

[0029] Six hydrogenase (EC 1.12.7.2) ORF sequences were identified in the genome of each of the acetogenic Clostridium species.

[0030] Twelve ferredoxin biosynthesis genes (Gene ID 40-51) were identified in the C. ragsdalei genome. Eleven ferredoxin biosynthesis genes (Gene ID 37-47, Table 2) were found in C. ljungdahlii, and twenty-six (Gene ID 36-61, Table 3) were found in C.

carboxidivorans.

[0031] Three genes coding for ferredoxin oxidoreductase enzymes were found in the C. ragsdalei genome that contain both a ferredoxin and nicotinamide cofactor binding domain. The ORF Sequence ID numbers (Table 1) for these genes are: RCCC02615; RCCC02028; and RCCC03071. The key gene for metabolic engineering, RCCC02028, is part of the cooS/cooF operon, also shown in FIG. 3. Similarly, three genes coding for ferredoxin oxidoreductase (FOR) enzymes were found in the C. ljungdahlii

genome. Each of these genes code for both the ferredoxin and cofactor binding domains. The ORF Sequence ID numbers for these genes are: RCCD00185;

RCCD01847; and RCCD00433 (Table 2). The key gene RCCD01847, is part of the cooF/cooS operon shown in FIG. 3.

[0032] Five genes were found in the C. carboxidivorans genome that contain both the ferredoxin and cofactor binding domains. The ORF Sequence ID numbers (Table 3) for these genes are: RCCB00442; RCCB01674; RCCB03510; RCCB00586; and RCCB 04795. The potentially key gene for modulating electron flow is RCCB03510, which is part of the cooF/cooS operon (FIG. 3).

[0033] The genes encoding AR (Gene ID 21, Table 1; Gene ID 19, Table 2) were sequenced in C. ragsdalei and C. ljungdahlii. A high degree of gene conservation is observed for the acetyl Co A reductase gene in C. ragsdalei and C.

ljungdahlii. Furthermore, in both micro-organisms, the enzyme exhibits a high degree of homology. The sequence of the acetyl CoA gene in c. ragsdalei and c. ljungdahlii was compared and found to have a 97.82% identity.

[0034] Further, the functionality of the gene (including the promoter) encoding for acetyl CoA reductase was tested. The gene was amplified by PCR, transferred into shuttle vector pCOS52 and ligated into the EcoRI site to form pCOS54. The vector contained the entire acetyl-CoA reductase gene and its promoter on a high-copy plasmid. pCOS52 contained the same backbone vector as pCOS54 but lacked the AR gene.

pCOS52 was used as the control plasmid in functional assays to determine expression of the AR gene in E. coli to confirm the Clostridial gene function. The results are shown in FIG.7, and confirm the function of the acetyl CoA reductase gene.

[0035] The functional assay consisted of adding cells harvested at the given time points to a reaction buffer containing NADPH and acetone as the substrate. Spectrophotometric activity (conversion of NADPH to NADP+) was measured at 378nm and compared to a standard curve to determine total activity level. Specific activity was determined using 317 mg/ gram of dry cell weight at an OD measurement of 1.

[0036] The genes encoding the PTA-ACK operon (Gene IDs 16-17, Tables 1 and 3; Gene IDs 15-16, Table 2) and its promoter were sequenced in C. ragsdalei, C. ljungdahlii, and C. carboxydivorans. The functionality of the operon was confirmed, and it was demonstrated that downregulation of the operon increases the ethanol to acetate production ratio. Downregulation involves decreasing the expression o the transcription of the 2-gene operon via promoter modification through site-directed mutagenesis. Such downregulation leads to a decrease in mRNA, leading to a decrease in protein production and a corresponding decrease in the ability of the strain to produce acetate. Such downregulation can be achieved via the method described in Example 2.

[0037] Additionally, a comparison was conducted of the genetic sequence found in the PTA-ACK operon across three species of acetogenic Clostridia. The PTA gene had 97% identity between c. ragsdalei and c. ljungdahlii, 78% identity between c. carboxydivorans and c. ragsdalei, and 79% identity between c. ljungdahlii and c. carboxydivorans. The ACK gene had 96% identity between c. ragsdalei and c. ljungdahlii, 78% between c. carboxydivorans and c. ragsdahlii, and 77% between c. carboxydivorans and c.

ljungdahlii. [0038] Key genes to promote production of ethanol in C. ragsdahlei include: SEQ ID NO l(Gene ID Nos. 4, 55, 53, Table 1) the gene sequence, including the experimentally determined promoter region, for carbon monoxide dehydrogenase, coos, electron transfer protein cooF, and the NADH dependent ferredoxin oxidoreductase (FOR) ;

SEQ ID NO 2 (Gene ID Nos. 17, 16, Table 1), the gene sequence, including the experimentally determined promoter reion, for ACK and PTA;

SEQ ID NO 3 (Gene ID No. 6, Table 1), the gene sequence, including the experimentally determined promoter region, for the acetyl CoA reductase;

Sequence Listing

C. ragsdalei gene sequences (Table 1)

>SEQ ID NO. 1 : (cooS, cooF, NADFLFerredoxin Oxidoreductase operon (includes STOP), Gene ID Nos. 4, 55, 53)

TATTATATCAATATAGAATAATTTTCAATCAAATAAGAATTATTTTATATTTT

ATATTGACAAGGAAACCGAAAAGGTTTATATTATTGTTATTGGATAACAATT

ATTTTTTAGTTAGTTGTACTTGTAAATAAATAGTATTAATTAATACTATTAAA

CTATTACAGTTTTTGATTCTTAGTATAAGTATTCTTAGTATCTTTAGCACTTAG

AATACGTTATCCTTTAGGAGAATAATCCTAATCAGTAATTTTAATAATTTAAT

AGTATACTTAAATAGTATAGTTTGGAGGTTTTATTATGTCAAATAACAAAATT

TGTAAGTCAGCAGATAAGGTACTTGAAAAGTTTATAGGTTCTCTAGATGGTGT

AGAAACTTCTCATCATAGGGTAGAAAGCCAAAGTGTTAAATGTGGTTTTGGT

CAGCTAGGAGTCTGCTGTAGACTCTGTGCAAACGGTCCCTGCAGAATAACAC

CTAAAGCTCCAAGAGGAGTATGTGGTGCTAGTGCTGATACCATGGTTGCAAG

AAACTTTCTTAGAGCTGTAGCTGCCGGCAGTGGATGTTATATCCATATAGTCG

AAAATACAGCTAGAAACGTAAAATCAGTAGGTGAAACCGGCGGAGAGATAA

AAGGAATGAATGCTCTCAACACCCTAGCAGAAAAACTTGGTATAACAGAATC

TGACCCACATAAAAAAGCTGTACTAGTAGCTGTGCCGTATTAAAGGACTTAT

ACAAACCAAAATTCGAAAAAATGGAAGTTATAAATAAATTAGCTTATGCACC

TAGACTAGAAAATTGGAACAAATTAAATATAATGCCTGGCGGTGCAAAATCA

GAAGTTTTTGATGGTGTAGTAAAAACTTCTACAAATCTAAACAGCGACCCTGT

AGATATGCTTCTAAATTGTTTAAAACTTGGAATATCCACTGGGATTTACGGAC

TTACCCTTACAAATTTATTAAATGACATAATTTTAGGTGAACCTGCTATAAGA

CCTGCAAAAGTTGGTTTTAAAGTTGTAGATACGGATTATATAAATTTGATGAT

AACAGGCCACCAGCACTCCATGATTGCCCACCTTCAAGAAGAACTTGTAAAA CCTGAAGCTGTAAAAAAAGCCCAAGCAGTTGGTGCTAAAGGATTCAAACTAG

TTGGATGTACCTGTGTCGGACAGGATTTACAGTTAAGAGGTAAATACTATACT

GATGTTTTCTCCGGTCATGCAGGAAATAACTTTACAAGTGAAGCCTTAATAGC

AACTGGAGGTATAGATGCAATAGTATCTGAATTTAACTGTACTCTTCCTGGCA

TCGAGCCAATAGCTGATAAGTTCATGGTTAAAATGATATGCCTAGATGACGT

TTCTAAAAAATCAAATGCAGAATATGTAGAATACTCTTTTAAAGATAGAGAA

AAAATAAGCAACCATGTTATAGATACGGCTATTGAAAGTTATAAGGAAAGAA

GATCTAAAGTTACAATGAATATTCCTAAAAACCATGGCTTTGATGACGTCATA

ACAGGTGTAAGTGAAGGTTCCTTAAAATCCTTCTTAGGCGGAAGTTGGAAAC

CTCTTGTAGACTTAATTGCTGCTGGAAAAATTAAAGGTGTTGCTGGAATAGTA

GGTTGTTCAAACTTAACTGCCAAAGGTCACGATGTATTTACAGTAGAACTTAC

AAAAGAACTCATAAAGAGAAATATAATTGTACTTTCTGCAGGTTGTTCAAGT

GGTGGACTTGAAAATGTAGGACTTATGTCTCCAGGAGCTGCTGAACTTGCAG

GAGATAGCTTAAAAGAAGTATGTAAGAGCCTAGGTATACCACCTGTACTAAA

TTTTGGTCCATGTCTTGCTATTGGAAGATTGGAAATTGTAGCAAAAGAACTAG

CAGAATACCTAAAAATAGATATTCCACAGCTTCCACTTGTGCTTTCTGCACCT

CAATGGCTTGAAGAACAAGCATTGGCAGATGGAAGTTTTGGTCTTGCCCTTG

GATTACCACTTCACCTTGCTATATCTCCTTTCATTGGTGGAAGCAAAGTGGTA

ACAAAAGTTTTATGTGAAGATATGGAAAATCTAACAGGCGGCAAGCTTATAA

TAGAAGACGATGTAATAAAAGCTGCAGATAAATTAGAAGAAACCATACTTGC

AAGAAGGAAAAGCTTAGGTCTTAATTAAATGAAAAGAATAATGATAAATAA

GGATTTATGTACCGGATGCTTAAATTGTACTTTAGCTTGTATGGCAGAACACA

ATGAAAATGGGAAATCTTTTTATGATCTGGATCTCAGCAATAAATTTCTTGAA

AGTAGAAATCATATATCTAAAGATGATAATGGAAACAAGCTTCCTATATTTT

GCCGTCACTGTGACGAACCTGAGTGCGTAATGACATGTATGAGCGGTGCCAT

GACTAAAGATCCTGAAACTGGTATAGTATCCTATGATGAGCATAAATGTGCC

AGCTGCTTTATGTGCGTCATGTCCTGTCCTTATGGAGTATTGAAACCAGATAC

TCAGACCAAAAGTAAAGTAGTTAAATGTGACCTGTGTGGTGACAGAGATACA

CCTAGATGCGTTGAAAATTGTCCAACAGAAGCAATTTATATTGAAAAGGAGG

CAGATCTCCTATGAATGAGTGGTTTAACAATAAAAATATTTTTTCACACAAAA

TATGTAATAATAGGAGCCAGTGCTGCTGGAATAAATGCTGCTAAAACTTTAA

GAAAGTTAGATAAATCCTCCAAAATAACTATTATTTCAAAGGATGATGCAGT

TTATTCAAGATGTATACTCCACAAAGTACTTGAGGGAAGTAGAAATTTAGAT

ACCATAAATTTTGTAGATTCTGATTTCTTTGAAAAAAATAATATAGAATGGAT

AAAAGATGCAGATGTAAGCAATATTGATATTGACAAGAAAAAAGTCTTACTT

CAAGACAACAGCAGCTTCAAATTTGACAAGCTCCTTATAGCTTCTGGTGCTTC

CTCCTTTATTCCCCCAGTTAAAAAATTAAGAGAAGCTAAAGGAGTGTACTCCC

TTAGAAATTTTGAAGATGTAACTGCTATACAAGACAAACTTAAAAACGCAAA

ACAAGTGGTAATACTTGGTGCAGGTCTTGTAGGAATTGATGCACTTTTAGGTC

TTATGGTGAAAAATATAAAGATTTCAGTTGTAGAAATGGGAGATAGGATTCT

CCCCCTTCAACTGGACAAAACTGCATCCACTATATATGAAAAGTTGTTAAAA

GAAAAAGGTATAGATGTCTTTACTTCAGTTAAATTGGAAGAGGTAGTTTTAA

ATAAAGACGGAACTGTAAGTAAAGCAGTACTATCAAATTCAACTTCTATAGA

TTGCGATATGATAATAGTTGCTGCTGGTGTTAGACCAAATGTAAGCTTTATAA

AAGACAGCAGGATAAAAGTTGAAAAAGGCATTGTCATAGACAAACATTGTA

AAACCACTGTAGATAATATATATGCTGCAGGAGATGTTACTTTTACTGCTCCT ATATGGCCTATAGCTGTAAAGCAGGGAATAACTGCTGCTTACAACATGGTAG

GTATAAATAGAGAATTACATGACACTTTTGGCATGAAGAACTCAATGAATTT

ATTTAACCTTCCATGCGTATCCCTTGGTAATGTAAATATAGCAGATGAAAGTT

ATGCTGTTGATACATTAGAAGGAGATGGAGTTTATCAAAAAATAGTTCACAA

AGATGGAGTAATCTACGGTGCACTTCTAGTTGGAGATATATCTTACTGCGGCG

TACTAGGATATCTCATAAAAAATAAAGTAAATATAAGCAATATCCATAAAAA

TATTTTTGACATAGATTATTCTGATTTTTACAATGTTGAAGAAGATGGACAAT

ATAGTTATCAATTGAGGTAA

SEQ ID NO. 2: (PTA-ACK operon (includes STOP), Gene ID Nos. 17, 16)

GCATACTGATTGATTATTTATTTGAAAATGCCTAAGTAAAATATATACATATT

ATAACAATAAAATAAGTATTAGTGTAGGATTTTTAAATAGAGTATCTATTTTC

AGATTAAATTTTTACTTATTTGATTTACATTGTATAATATTGAGTAAAGTATTG

ACTAGTAAAATTTTGTGATACTTTAATCTGTGAAATTTCTTAGCAAAAGTTAT

ATTTTTGAATAATTTTTATTGAAAAATACAACTAAAAAGGATTATAGTATAAG

TGTGTGTAATTTTGTGTTAAATTTAAAGGGAGGAAATAAACATGAAATTGAT

GGAAAAAATTTGGAATAAGGCAAAGGAAGACAAAAAAAAGATTGTCTTAGC

TGAAGGAGAAGAAGAAAGAACTCTTCAAGCTTGTGAAAAAATAATTAAAGA

AGGTATTGCAAATTTAATCCTTGTAGGGAATGAAAAGGTAATAGAGGAGAAG

GCATCAAAATTAGGCGTAAGTTTAAATGGAGCAGAAATAGTAGATCCAGAAA

CCTCGGATAAACTAAAAAAATATGCAGATGCTTTTTATGAATTGAGAAAGAA

GAAGGGAATAACACCAGAAAAAGCGGATAAAATAGTAAGAGATCCAATATA

TTTTGCTACGATGATGGTTAAGCTTGGAGATGCAGATGGATTGGTTTCAGGTG

CAGTGCATACTACAGGTGATCTTTTGAGACCAGGACTTCAAATAGTAAAGAC

AGCTCCAGGTACATCAGTAGTTTCCAGCACATTTATAATGGAAGTACCAAATT

GTGAATATGGTGACAATGGTGTACTTCTATTTGCTGATTGTGCTGTAAATCCA

TGCCCAGATAGTGATCAATTGGCTTCAATTGCAATAAGTACAGCAGAAACTG

CAAAGAACTTATGTGGAATGGATCCAAAAGTAGCAATGCTTTCATTTTCTACT

AAGGGAAGTGCAAAACACGAATTAGTAGATAAAGTTAGAAATGCTGTAGAA

ATTGCCAAAAAAGCTAAACCAGATTTAAGTTTGGACGGAGAATTACAATTAG

ATGCCTCTATCGTAGAAAAGGTTGCAAGTTTAAAGGCTCCTGAAAGTGAAGT

AGCAGGAAAAGCAAATGTACTTGTATTTCCAGATCTCCAAGCAGGAAATATA

GGTTATAAACTTGTTCAAAGATTTGCAAAAGCTGATGCTATAGGACCTGTATG

CCAGGGATTTGCAAAACCTATAAATGATTTGTCAAGAGGATGTAACTCCGAT

GATATAGTAAATGTAGTAGCTGTAACAGCAGTTCAGGCACAAGCTCAAAAGT

AAATGAAAATATTAGTAGTAAACTGTGGAAGTTCATCTTTAAAATATCAACTT

ATTGATATGAAAGATGAAAGCGTTGTGGCAAAAGGACTTGTAGAAAGAATA

GGAGCAGAAGGTTCAGTTTTAACACATAAAGTTAACGGAGAAAAGTTTGTTA

CAGAGCAGCCAATGGAAGATCATAAAGTTGCTATACAATTAGTATTAAATGC

TCTTGTAGATAAAAAACATGGTGTAATAAAAGATATGTCAGAAATATCTGCT

GTAGGGCATAGAGTTTTGCATGGTGGAAAAAAATATGCGGCATCCATTCTTA

TTGATGACAATGTAATGAAAGCAATAGAAGAATGTATTCCATTAGGACCATT

ACATAATCCAGCTAATATAATGGGAATAGATGCTTGTAAAAAACTAATGCCA

AATACTCCAATGGTAGCAGTATTTGATACAGCATTTCATCAGACAATGCCAG

ATTATGCTTATACTTATGCAATACCTTATGATATATCTGAAAAGTATGATATC

AGAAAATATGGTTTTCATGGAACTTCTCATAGATTCGTTTCAATTGAAGCAGC CAAGTTGTTAAAGAAAGATCCAAAAGATCTTAAGCTAATAACTTGTCATTTA

GGAAATGGAGCTAGTATATGTGCAGTAAACCAGGGAAAAGCAGTAGATACA

ACTATGGGACTTACTCCCCTTGCAGGACTTGTAATGGGAACTAGATGTGGTG

ATATAGATCCAGCTATAATACCATTTGTAATGAAAAGAACAGGTATGTCTGT

AGATGAAATGGATACTTTAATGAACAAAAAGTCAGGAATACTTGGAGTATCA

GGAGTAAGCAGCGATTTTAGAGATGTAGAAGAAGCTGCAAATTCAGGAAAT

GATAGAGCAAAACTTGCATTAAATATGTATTATCACAAAGTTAAATCTTTCAT

AGGAGCTTATGTTGCAGTTTTAAATGGAGCAGATGCTATAATATTTACAGCA

GGACTTGGAGAAAATTCAGCTACTAGCAGATCTGCTATATGTAAGGGATTAA

GCTATTTTGGAATTAAAATAGATGAAGAAAAGAATAAGAAAAGGGGAGAAG

CACTAGAAATAAGCACACCTGATTCAAAGATAAAAGTATTAGTAATTCCTAC

AAATGAAGAACTTATGATAGCTAGGGATACAAAAGAAATAGTTGAAAATAA

ATAA

SEQ ID NO. 3: (ORF RCCC02715, PI 1, NADPH-SADH (includes STOP), Gene ID No. 6)

ATGAAAGGTTTTGCAATGTTAGGTATTAACAAGTTAGGATGGATTGAAAAGA

AAAACCCAGTACCAGGTCCTTATGATGCGATTGTACATCCTCTAGCTGTATCC

CCATGTACATCAGATATACATACGGTTTTTGAAGGAGCACTTGGTAATAGGG

AAAATATGATTTTAGGTCACGAAGCTGTAGGTGAAATAGCTGAAGTTGGCAG

TGAAGTTAAAGATTTTAAAGTTGGCGATAGAGTTATCGTACCATGCACAACA

CCTGACTGGAGATCCTTAGAAGTCCAAGCTGGTTTTCAACAGCATTCAAACG

GTATGCTTGCAGGATGGAAGTTTTCCAATTTTAAAGACGGTGTATTTGCAGAT

TACTTTCATGTAAACGATGCAGATATGAATCTTGCAATACTTCCAGATGAAAT

ACCTTTAGAAAGTGCAGTTATGATGACAGACATGATGACTACTGGTTTTCATG

GGGCAGAACTTGCTGACATAAAAATGGGTTCCAGTGTTGTCGTAATTGGTAT

AGGAGCTGTTGGATTAATGGGAATAGCCGGTTCCAAACTTCGAGGAGCAGGT

AGAATTATCGGTGTTGGAAGCAGACCCGTTTGTGTTGAAACAGCTAAATTTTA

TGGAGCAACTGATATTGTAAATTATAAAAATGGTGATATAGTTGAACAAATA

ATGGACTTAACTCATGGTAAAGGTGTAGACCGTGTAATCATGGCAGGCGGTG

GTGCTGAAACACTAGCACAAGCAGTAACTATGGTTAAACCTGGCGGCGTAAT

TTCTAACATCAACTACCATGGAAGCGGTGATACTTTGCCAATACCTCGTGTTC

AATGGGGCTGCGGCATGGCTCACAAAACTATAAGAGGAGGGTTATGTCCCGG

CGGACGTCTTAGAATGGAAATGCTAAGAGACCTTGTTCTATATAAACGTGTT

GATTTGAGCAAACTTGTTACTCATGTATTTGATGGTGCAGAAAATATTGAAAA

GGCCCTTTTGCTTATGAAAAATAAGCCAAAAGATTTAATTAAATCAGTAGTTA

CATTCTAA

[0039] Using detailed genomic information, the acetogenic Clostridia micro-organisms have been metabolically engineered to increase the carbon and electron flux through the biosynthetic pathways for ethanol and butanol, while simultaneously reducing or eliminating carbon and electron flux through the corresponding acetate and butyrate formation pathways, in accordance with the present invention. For this purpose, the activities of key genes encoding for enzymes in the pathway have been modulated. In one embodiment, gene expression of key alcohol producing enzymes is increased by increasing the copy number of the gene. For example, a key carbon monoxide dehydrogenase operon (FIG.3) and the associated electron transfer proteins, including acetyl CoA reductase and aldehyde ferredoxin oxidoreductase are duplicated within the genome of the modified organism. In one embodiment, these duplications are introduced into strains having knocked out or attenuated acetate production to further channel electrons into the ethanol or butanol production pathway. In another embodiment a knockout strategy is applied to strains of acetogenic Clostridia that, when grown on syngas, produce more complex mixtures of alcohols and acids, such as ethanol, butanol and hexanol and their corresponding carboxylic acids.

[0040] In one embodiment, vectors to be used for the transfer of acetogenic Clostridia cloned genes from cloning vehicles to parent acetogenic Clostridia strains are constructed using standard methods (Sambrook et al., 1989). All gene targets used in molecular genetics experiments are amplified using high-fidelity polymerase chain reaction (PCR) techniques using sequence-specific primers. The amplified genes are next subcloned into intermediate cloning vehicles, and later recombined in multi-component ligation reactions to yield the desired recombinant vector to be used in the gene transfer experiments. The vectors contain the appropriate functional features required to carry out the gene transfer experiments successfully and vary depending on the method used.

[0041] To transfer the recombinant vectors into recipient acetogenic Clostridia, a variety of methods are used. These include electroporation, bi-parental or tri-parental conjugation, liposome-mediated transformation and polyethylene glycol-mediated transformation. Recombinant acetogenic Clostridia are isolated and confirmed through molecular biology techniques based on the acquisition of specific traits gained upon DNA integration.

[0042] Example 1

[0043] Acetogenic Clostridia contain operon 300, shown in FIG. 3, that consists of carbon monoxide dehydrogenase 104(cooS, Gene ID 4, Table 1, Table 2, Table 3), a membrane-associated electron transfer protein (cooF), and a ferredoxin oxidoreductase (FOR). Overexpression of carbon monoxide dehydrogenase 104 within the acetogenic Clostridia is known to increase electron flow from syngas components to the oxidizeded nucleotide cofactors NAD ⁺ and NADP ⁺ The increased levels of reduced nucleotide cofactors then stimulate generation of intermediate compounds in Wood-Ljungdahl pathway 100.

[0044] In one embodiment, operon 300 is amplified using long-PCR techniques with primers that are designed to anneal to a region 200 nucleotides (nt) upstream of the carbon monoxide dehydrogenase gene and 200 nt downstream of the ferredoxin oxidoreductase gene. The total region is about 3.8 kilobase pairs. The amplified DNA is cloned directly into suitable plasmid vectors specifically designed to ligate PCR products such as pGEM T easy (Promega, Madison, WI) or pTOPO (Invitrogen, Carlsbad, CA). The ends of the PCR product contain engineered restriction sites to facilitate later cloning steps. The operon 300 is subcloned into a vector that already contains cloned chromosomal C. ragsdalei or other acetogenic Clostridial DNA to allow chromosomal integration at a neutral site.

[0045] Example 2

[0046] Because carboxylic acids compete with alcohols for electrons, decreasing acid production allows more electrons to flow down the alcohol-production pathway from the CoA intermediate directly to the alcohol. Acetogenic Clostridia contain genes for phospho-transacetylase enzyme (Gene ID 17, Tables 1 and 3; Gene ID 16, Table 2) that converts acetyl-CoA to acetyl-phosphate and acetate kinase (Gene ID 16, Table 1) that converts acetyl-phosphate 218 to acetate 214. In one embodiment, genetic modifications to delete all or part of the genes for both enzymes and knock out or attenuate production of acetate are made as shown in FIG. 4.

[0047] Using PCR and other standard methods, a recombinant vector containing two large non-contiguous segments of DNA is generated. Upon replacement of the native gene by the recombinant vector gene, the Clostridial strain contains no

phoshotransacetylase or acetate kinase activities as shown in FIG 4 by X 504 and X 502, respectively.

[0048] Modulation of the common promoter region, P* 506 to attenuate gene expression of phosphotransacetylase 508 and acetate kinase 510 and subsequent acetate production are carried out by generating a series of recombinant vectors with altered promoter regions. The vector series is constructed by site-directed mutagenesis.

[0049] Additionally, down-regulation of the 2-gene operon containing pta/ack genes is performed by site-directed mutagenesis of the promoter region. A decrease in RNA polymerase binding leads to a decrease in transcriptional activity off of the pta/ack promoter and in turn lead to a decrease in protein activity. The end result is a decrease in acetate production since the intermediates are produced at a lower rate and more carbon from acetyl-CoA goes towards ethanol production. A promoter probe assay using a reporter group that is easily quantitated has been developed to measure relative promoter strength of the pta/ack promoter in vivo. After site-directed mutagenesis is performed, which imparts single and multiple lesions over a 200 base pair region, strains that have decreased promoter activity are isolated such that a series of strains with 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% and 0% activity of the native promoter in the assay are isolated and tested in recombinant Clostridia strains.

[0050] Example 3

[0051] In vivo, the acetyl Co A enzyme designated in 102 and FIG 4 converts the Coenzyme A (CoA) form of a carbon moiety, such as acetyl-CoA 102 or butyrl-CoA directly to its corresponding alcohol. Thermodynamically, direct conversion from the CoA form to the alcohol requires transfer of four electrons, and is a more efficient way to generate the alcohol, compared to the two-step conversion of the carboxylic acid to the corresponding alcohol. For example, as shown in FIG 5, the two step conversion requires that acetate 214, first be converted to its aldehyde form (acetaldehyde, 604), and then to the corresponding alcohol, ethanol 216. Thus, increasing AR activity, portrayed by the vertical arrow 602 is desirable for increasing alcohol production, and increasing the selectivity of the process by increasing the ratio of alcohol to acid.

[0052] In one embodiment, AR activity in acetogenic Clostridia is increased by amplifying the gene in vitro using high-fidelity PCR and inserting the duplicated copy of the gene into a neutral site in the chromosome using standard molecular genetic techniques. After gene replacement of the vector, the chromosome contains two copies of the AR. Confirmation of genereplacement followed by gene expression studies of the recombinant strain are performed and compared to the parent strain.

[0053] In other embodiments a similar strategy is used to increase the enzymatic activity of adhE-type alcohol dehydrogenases, short-chain alcohol-dehydrogenases and primary Fe-containing alcohol dehydrogenases.

[0054] Example 4

[0055] Under some conditions, Clostridia need to obtain additional energy in the form of adenosine triphosphate production (ATP) causing the cells to temporarily increase the production of acetate 214 from acetyl-CoA 102. The net reaction is 1 ATP from ADP + Pi through acetyl-phosphate. Acetate production is advantageous to the syngas

fermentation process at low to moderate acetic acid concentrations, because it allows the cells to produce more energy and remain robust. However, too much free acetic acid causes dissipation of the transmembrane ion gradient used as the primary ATP generation source and therefore becomes detrimental to the cells. For industrial production purposes, it is advantageous to convert the acetate to ethanol to increase ethanol production and reduce the probability of accumulating too much free acetic acid.

[0056] In one embodiment, ethanol production in the double mutant C. ragsdahlei strain is increased by between 10 and 40% as a result of the increased aldehyde ferredoxin oxidoreductase and AR activities. In another embodiment, the ratio of ethanol to acetate produced is increased between 5 and 10 fold, but allows sufficient acetate formation to support ATP production needed to meet the energy needs of the microorgansim.

[0057] While the invention has been described with reference to particular

embodiments, it will be understood by one skilled in the art that variations and modifications may be made in form and detail without departing from the spirit and scope of the invention.