Patent Abstract:
Recombinant acetogenic Clostridia are engineered to modulate production of aliphatic C 2 -C 6  alcohols and aliphatic C 2 -C 6  organic acids from synthetic gases. One aspect of the invention provides a method of producing an aliphatic C 2 -C 6  alcohol using an acetogenic  Clostridium  micro-organism having at least one genetic modification that reduces or eliminates C 2 -C 6  carboxylic acid production by the modified organism. Another aspect of the invention provides a method of producing an aliphatic C 2 -C 6  alcohol using an acetogenic  Clostridium  micro-organism having one or more genetic modifications that cause increased enzyme activity of carbon monoxide dehydrogenase, aldehyde ferredoxin oxidoreductase, NADPH-dependent alcohol dehydrogenase, or alcohol dehydrogenase. Yet another aspect of the invention provides a method of producing aliphatic C2-C6 alcohols using acetogenic  Clostridium  micro-organisms that have been genetically modified to increase C 2 -C 6  aliphatic alcohol formation and decrease the production of the corresponding C 2 -C 6  aliphatic organic acid.

Full Description:
TECHNICAL FIELD 
     This invention relates generally to metabolically engineering microorganisms that use the Wood-Ljundahl pathway to produce aliphatic C 2 -C 6  alcohols. More specifically, the invention relates to engineering recombinant acetogenic Clostridia to modulate production of aliphatic C 2 -C 6  alcohols and aliphatic C 2 -C 6  organic acids from synthesis gases. 
     BACKGROUND OF THE INVENTION 
     Synthesis gas (syngas) is a mixture of carbon monoxide (CO) gas, carbon dioxide (CO 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. Acetogenic Clostridia microorganisms grown in an atmosphere containing syngas are  c apable of absorbing th e  syngas components CO, CO 2 , and H 2  and pro d uci n g al i pha t ic 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-CoA 102, a key intermediate in the pathway. The enzymes activating Wood-Ljundahl pathway 100 are carbon monoxide dehydrogenase (CODH) 104 a n d hydro g enase (H 2 ase) 106. These  e nz y mes capture the electrons fr o m 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 b e tween −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 nucleotide cofactors (NAD +  and NADP + ) are used for the generation of intermediate compounds in Wood-Ljungdahl pathway 100 leading to acetyl-CoA 102 formation. 
     Acetyl-CoA 102 formation t hr ough W oo d-Ljungdahl pathway 100 is shown in greater detail in  FIG. 2 . Eit h er CO 2  202 or  C O 208 provide substrates for the pathway. The carbon from CO 2  202 is reduced to a methyl group t h rough suc c essive 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 -C 6  alcohol s and acids in acetogenic Clostridia. 
     Wood-Ljungdahl pathway 100 is neutral with respect to ATP production when acetate 214 is produced ( FIG. 2 ). When ethanol 216 is produced directly from acetyl-CoA 102, 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-PO 4  218 is converted to acetate 214. 
     Acetogenic Clostridia organisms generate cellular energy by ion gradient-driven phophorylation. 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 dehydrogenase, 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. 
     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, could be lowered significantly if the production could be maximized by reducing or eliminating production of the corresponding acid, in this example acetate. Under some fermentation conditions, the production of acetate is best maintained within a range in which sufficient acetyl-PO 4  is converted to acetate to produce adequate ATP for the micro-organisms&#39; viability without accumulation of acetic acid. It would therefore be desirable to metabolically engineer acetogenic Clostridia for improved production of selected C 2 -C 6  alcohol s or acids through Wood-Ljungdahl pathway 100 by modulating enzymatic activities of key enzymes in the pathway. 
     SUMMARY OF THE INVENTION 
     One aspect of the invention provides a method of producing an aliphatic C 2 -C 6  alcohol by providing an acetogenic  Clostridium  micro-organism having a genetic modification within at least one transcriptional unit that substantially reduces C 2 -C 6  carboxylic acid production by the organism. A growth medium and a syngas are supplied, and the modified acetogenic  Clostridium  micro-organism is cultured in the growth medium in the presence of the syngas to produce the aliphatic C 2 -C 6  alcohol. 
     Another aspect of the invention provides a method of producing an aliphatic C 2 -C 6  alcohol by providing an acetogenic  Clostridium  micro-organism having a genetic modification within at least one transcriptional unit that increases aliphatic C 2 -C 6  alcohol production by the micro-organism. A growth medium and a syngas are supplied, and the modified acetogenic  Clostridium  micro-organism is cultured in the growth medium in the presence of the syngas to produce the aliphatic C 2 -C 6  alcohol. 
     Another aspect of the invention provides a method of producing an aliphatic C 2 -C 6  alcohol by providing a  Clostridium  micro-organism having at least two transcriptional units. A genetic modification within the first transcriptional unit substantially reduces aliphatic C 2 -C 6  carboxylic acid production, and a second genetic modification within the second transcriptional unit substantially increases aliphatic C 2 -C 6  alcohol production by the micro-organism. A growth medium and a syngas are supplied, and the modified acetogenic  Clostridium  micro-organism is cultured in the growth medium in the presence of the syngas to produce the aliphatic C 2 -C 6  alcohol. 
     Yet another aspect of the invention provides a method of producing butanol by providing  Clostridium carboxidivorans  having at least one genetic modification that reduces or eliminates butyrate production by the organism. A growth medium and a syngas are supplied, and the modified  Clostridium carboxidivroans  organism is cultured in the growth medium in the presence of the syngas to produce butanol. 
     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 
         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; 
         FIG. 2  is a diagram illustrating the Wood-Ljungdahl (C 1 ) pathway for acetyl-CoA production and the enzymatic conversion of acetyl-CoA to acetate and ethanol; 
         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; 
         FIG. 4  is a diagram showing alignment of the genome architecture via the protein encoded by the gene for NADPH dependent secondary alcohol dehydrogenase in  C. ragsdalei  (SEQ ID NO:33),  C. ljungdahlii  (SEQ ID NO:34) and Thermoanaerobactor ethanolicus (SEQ ID NO:35), in accordance with the invention; 
         FIG. 5  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 modifying (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; 
         FIG. 6  is a diagram of the Wood-Ljungdahl pathway for ethanol synthesis, and showing a strategy for specifically increasing ethanol production in  C. ragsdahlei  by overexpression of a NADPH-dependent alcohol dehydrogenase in a host knocked out for acetate kinase or phosphotransacetylase activity, in accordance with the invention; 
         FIG. 7  is a diagram of the Wood-Ljungdahl pathway for ethanol synthesis, and showing a strategy for increasing ethanol production in acetogenic Clostridia by specifically increasing the conversion rate of acetate to ethanol through overexpression of aldehyde ferredoxin oxidoreductase (AOR) in a host strain that is attenuated in its ability to produce acetate and has increased NADPH-dependent alcohol dehydrogenase activity, in accordance with the invention; and 
         FIG. 8  is a diagram of the butanol and butyrate biosynthesis pathway in  C. carboxidivorans  and the corresponding genes catalyzing the conversion of acetyl-CoA to butanol and butyrate showing a strategy for increasing butanol production, in accordance with the invention. 
     
    
    
     DETAILED DESCRIPTION 
     The present invention is directed to acetogenic Clostridia micro-organisms that have been genetically modified to increase production of aliphatic C 2 -C 6  alcohols. Alcohol production can be increased by either increasing expression of a transcriptional unit that increases alcohol production or decreasing expression of a transcriptional unit needed for production of a C 2 -C 6  carboxylic acid. A transcriptional unit is either a gene, a single unit, that codes for one enzyme and is controlled by a promoter, or an operon, a multiple transcriptional unit, that codes for two or more genes, and whose total gene expression is controlled by the same promoter. For example alcohol production can be increased by either increasing transcription of the gene that codes for NADPH-dependent alcohol dehydrogenase, or by reducing or eliminating transcription of the operon that codes for acetate kinase and phosphotransacetylase, and by this means, reducing production of the corresponding C 2 -C 6  organic acid. 
     Several species of acetogenic Clostridia that produce C 2 -C 6  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 micro-organisms were sequenced in order to locate and modify the portions of the genome that code for the enzymes of interest. 
     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 . 
     
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                   Clostridium ragsdalei  genes used in metabolic engineering. 
               
             
          
           
               
                 Pathway 
                 Gene ID 
                 Gene Name 
                 EC number 
                 ORF ID 
                 Copy ID 
                 Description 
               
               
                   
               
             
          
           
               
                 Wood- 
                 1 
                 Carbon Monoxide 
                 1.2.2.4 
                 RCCC00183 
                 CODH_1 
                 CO oxidation 
               
               
                 Ljungdahl 
                 2 
                 Dehydrogenase 
                   
                 RCCC01175 
                 CODH_2 
                 CO oxidation 
               
               
                   
                 3 
                   
                   
                 RCCC01176 
                 CODH_3 
                 CO oxidation 
               
               
                   
                 4 
                   
                   
                 RCCC02026 
                 CODH_4 
                 CO oxidation 
               
               
                   
                 5 
                   
                   
                 RCCC03874 
                 CODH_5 
                 CO oxidation 
               
               
                   
                 6 
                 Carbon Monoxide 
                 1.2.99.2 
                 RCCC03862 
                 cooS/acsA 
                 bifunctional 
               
               
                   
                   
                 Dehydrogenase/Acetyl- 
                   
                   
                   
                 CODH/ACS 
               
               
                   
                   
                 CoA Synthase 
                   
                   
                   
                 enzyme, carbon 
               
               
                   
                   
                   
                   
                   
                   
                 fixation 
               
               
                   
                 7 
                 Formate Dehydrogenase 
                 1.2.1.2 
                 RCCC00874 
                 FDH_1 
                 Methyl branch 
               
               
                   
                 8 
                   
                   
                 RCCC03324 
                 FDH_2 
                 carbon fixation 
               
               
                   
                 9 
                 Formyltetrahydrofolate 
                 6.3.4.3 
                 RCCC03872 
                 FTHFS 
                 Methyl branch 
               
               
                   
                   
                 Synthase 
                   
                   
                   
                 carbon fixation 
               
               
                   
                 10 
                 Methenyltetrahydrofolate 
                 3.5.4.9 
                 RCCC03870 
                 MEC 
                 Methyl branch 
               
               
                   
                   
                 cyclohydrolase 
                   
                   
                   
                 carbon fixation 
               
               
                   
                 11 
                 Methylenetetrahydrofolate 
                 1.5.1.5 
                 RCCC03870 
                 MED 
                 Methyl branch 
               
               
                   
                   
                 dehydrogenase 
                   
                   
                   
                 carbon fixation 
               
               
                   
                 12 
                 Methylenetetrahydrofolate 
                 1.5.1.20 
                 RCCC03868 
                 MER 
                 Methyl branch 
               
               
                   
                   
                 reductase 
                   
                   
                   
                 carbon fixation 
               
               
                   
                 13 
                 Methyltransferase 
                 2.1.1.13 
                 RCCC03863 
                 acsE 
                 Methyl branch 
               
               
                   
                   
                   
                   
                   
                   
                 carbon fixation 
               
               
                   
                 14 
                 Corrinoid/Iron-sulfur 
                 1.2.99.2 
                 RCCC03864 
                 acsC 
                 Part of 
               
               
                   
                   
                 protein 
                   
                   
                   
                 CODH/ACS 
               
               
                   
                   
                   
                   
                   
                   
                 complex, Large 
               
               
                   
                   
                   
                   
                   
                   
                 subunit 
               
               
                   
                 15 
                 Corrinoid/Iron-sulfur 
                 1.2.99.2 
                 RCCC03865 
                 acsD 
                 Part of 
               
               
                   
                   
                 protein 
                   
                   
                   
                 CODH/ACS 
               
               
                   
                   
                   
                   
                   
                   
                 complex, Small 
               
               
                   
                   
                   
                   
                   
                   
                 subunit 
               
               
                 Ethanol and 
                 16 
                 Acetate Kinase 
                 2.7.2.1 
                 RCCC01717 
                 ACK 
                 Acetate 
               
               
                 acetate 
                   
                   
                   
                   
                   
                 production 
               
               
                 production 
                 17 
                 Phospho-transacetylase 
                 2.3.1.8 
                 RCCC01718 
                 PTA 
                 Acetate 
               
               
                   
                   
                   
                   
                   
                   
                 production 
               
               
                   
                 18 
                 Tungsten-containing 
                 1.2.7.5 
                 RCCC00020 
                 AOR_1 
                 Reduction of 
               
               
                   
                   
                 aldehyde ferredoxin 
                   
                   
                   
                 acetate to 
               
               
                   
                   
                 oxidoreductase 
                   
                   
                   
                 acetaldehyde 
               
               
                   
                 19 
                   
                 1.2.7.5 
                 RCCC00030 
                 AOR_2 
                 Reduction of 
               
               
                   
                   
                   
                   
                   
                   
                 acetate to 
               
               
                   
                   
                   
                   
                   
                   
                 acetaldehyde 
               
               
                   
                 20 
                   
                 1.2.7.5 
                 RCCC01183 
                 AOR_3 
                 Reduction of 
               
               
                   
                   
                   
                   
                   
                   
                 acetate to 
               
               
                   
                   
                   
                   
                   
                   
                 acetaldehyde 
               
               
                   
                 21 
                 Alcohol Dehydrogenase 
                 1.1.1.2 
                 RCCC02715 
                 ADH_1 
                 zinc containing, 
               
               
                   
                   
                   
                   
                   
                   
                 NADPH 
               
               
                   
                   
                   
                   
                   
                   
                 dependent 
               
               
                   
                   
                   
                   
                   
                   
                 secondary ADH 
               
               
                   
                 22 
                   
                 1.1.1.1 
                 RCCC01356 
                 ADH_2 
                 two pfam domain: 
               
               
                   
                   
                   
                   
                   
                   
                 FeADH and 
               
               
                   
                   
                   
                   
                   
                   
                 ALDH, AdhE 
               
               
                   
                 23 
                   
                 1.1.1.1 
                 RCCC01357 
                 ADH_3 
                 two pfam domain: 
               
               
                   
                   
                   
                   
                   
                   
                 FeADH and 
               
               
                   
                   
                   
                   
                   
                   
                 ALDH, AdhE 
               
               
                   
                 24 
                   
                 1.1.1.1 
                 RCCC01358 
                 ADH_4 
                 two pfam domain: 
               
               
                   
                   
                   
                   
                   
                   
                 FeADH and 
               
               
                   
                   
                   
                   
                   
                   
                 ALDH, AdhE, 
               
               
                   
                   
                   
                   
                   
                   
                 fragment (76aa) 
               
               
                   
                 25 
                   
                 1.1.1.1 
                 RCCC03300 
                 ADH_5 
                 one pfam domain: 
               
               
                   
                   
                   
                   
                   
                   
                 FeADH 
               
               
                   
                 26 
                   
                 1.1.1.1 
                 RCCC03712 
                 ADH_6 
                 one pfam domain: 
               
               
                   
                   
                   
                   
                   
                   
                 FeADH 
               
               
                   
                 27 
                   
                 1.1.1.1 
                 RCCC04095 
                 ADH_7 
                 one pfam domain: 
               
               
                   
                   
                   
                   
                   
                   
                 FeADH 
               
               
                   
                 28 
                   
                 1.—.—.— 
                 RCCC00004 
                 ADH_8 
                 short chain ADH, 
               
               
                   
                   
                   
                   
                   
                   
                 multiple copy 
               
               
                   
                 29 
                   
                 1.—.—.— 
                 RCCC01567 
                 ADH_9 
                 short chain ADH, 
               
               
                   
                   
                   
                   
                   
                   
                 multiple copy 
               
               
                   
                 30 
                   
                 1.—.—.— 
                 RCCC02765 
                 ADH_10 
                 short chain ADH, 
               
               
                   
                   
                   
                   
                   
                   
                 multiple copy 
               
               
                   
                 31 
                   
                 1.—.—.— 
                 RCCC02240 
                 ADH_11 
                 short chain ADH, 
               
               
                   
                   
                   
                   
                   
                   
                 multiple copy 
               
               
                   
                 32 
                 Aldehyde 
                 1.2.1.10 
                 RCCC03290 
                 ALDH_1 
                 Acetylating 
               
               
                   
                 33 
                 Dehydrogenase 
                 1.2.1.10 
                 RCCC04101 
                 ALDH_2 
                 Acetylating 
               
               
                   
                 34 
                   
                 1.2.1.10 
                 RCCC04114 
                 ALDH_3 
                 Acetylating 
               
               
                 Hydrogenase 
                 35 
                 Hydrogenase 
                 1.12.7.2 
                 RCCC00038 
                 HYD_1 
                 Fe only, H2 
               
               
                   
                   
                   
                   
                   
                   
                 production 
               
               
                   
                 36 
                   
                 1.12.7.2 
                 RCCC00882 
                 HYD_2 
                 Fe only, large 
               
               
                   
                   
                   
                   
                   
                   
                 subunit, H2 
               
               
                   
                   
                   
                   
                   
                   
                 production 
               
               
                   
                 37 
                   
                 1.12.7.2 
                 RCCC01252 
                 HYD_3 
                 Fe only, H2 
               
               
                   
                   
                   
                   
                   
                   
                 production 
               
               
                   
                 38 
                   
                 1.12.7.2 
                 RCCC01504 
                 HYD_4 
                 Fe only, H2 
               
               
                   
                   
                   
                   
                   
                   
                 production 
               
               
                   
                 39 
                   
                 1.12.7.2 
                 RCCC02997 
                 HYD_5 
                 Ni—Fe large 
               
               
                   
                   
                   
                   
                   
                   
                 subunit, H2 
               
               
                   
                   
                   
                   
                   
                   
                 oxidation 
               
               
                 Electron 
                 40 
                 Ferredoxin 
                   
                 RCCC00086 
               
               
                 carrier 
                 41 
                   
                   
                 RCCC00301 
               
               
                   
                 42 
                   
                   
                 RCCC00336 
               
               
                   
                 43 
                   
                   
                 RCCC01168 
               
               
                   
                 44 
                   
                   
                 RCCC01415 
               
               
                   
                 45 
                   
                   
                 RCCC01825 
               
               
                   
                 46 
                   
                   
                 RCCC02435 
               
               
                   
                 47 
                   
                   
                 RCCC02890 
               
               
                   
                 48 
                   
                   
                 RCCC03063 
               
               
                   
                 49 
                   
                   
                 RCCC03726 
               
               
                   
                 50 
                   
                   
                 RCCC04003 
               
               
                   
                 51 
                   
                   
                 RCCC04147 
               
               
                 Electron 
                 52 
                 Pyridine nucleotide- 
                   
                 RCCC02615 
                   
                 glutamate 
               
               
                 transfer 
                   
                 disulphide 
                   
                   
                   
                 synthase small 
               
               
                   
                   
                 oxidoreductases 
                   
                   
                   
                 chain, but no large 
               
               
                   
                   
                   
                   
                   
                   
                 chain next to it 
               
               
                   
                 53 
                   
                   
                 RCCC02028 
                   
                 next to cooF and 
               
               
                   
                   
                   
                   
                   
                   
                 cooS, probably 
               
               
                   
                   
                   
                   
                   
                   
                 important for 
               
               
                   
                   
                   
                   
                   
                   
                 reduced pyridine 
               
               
                   
                   
                   
                   
                   
                   
                 cofactor 
               
               
                   
                   
                   
                   
                   
                   
                 generation 
               
               
                   
                 54 
                   
                   
                 RCCC03071 
                   
                 NADH 
               
               
                   
                   
                   
                   
                   
                   
                 dehydrogenase, 
               
               
                   
                   
                   
                   
                   
                   
                 not part of an 
               
               
                   
                   
                   
                   
                   
                   
                 operon 
               
               
                   
                 55 
                 Membrane-associated 
                   
                 RCCC02027 
                 cooF 
                 Between gene 
               
               
                   
                   
                 electron transfer FeS 
                   
                   
                   
                 number 4 and 
               
               
                   
                   
                 protein, cooF 
                   
                   
                   
                 gene number 53 
               
               
                   
               
             
          
         
       
     
     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. 
     
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                   Clostridium ljungdahlii  genes used in metabolic engineering. 
               
             
          
           
               
                 Pathway 
                 Gene ID 
                 Gene Name 
                 EC number 
                 ORE ID 
                 Copy ID 
                 Description 
               
               
                   
               
             
          
           
               
                 Wood- 
                 1 
                 Carbon Monoxide 
                 1.2.2.4 
                 RCCD00983 
                 CODH_1 
                 CO oxidation 
               
               
                 Ljungdahl 
                 2 
                 Dehydrogenase 
                   
                 RCCD00984 
                 CODH_2 
                 CO oxidation 
               
               
                   
                 3 
                   
                   
                 RCCD01489 
                 CODH_3 
                 CO oxidation 
               
               
                   
                 4 
                   
                   
                 RCCD04299 
                 CODH_4 
                 CO oxidation 
               
               
                   
                 5 
                 Carbon Monoxide 
                 1.2.99.2 
                 RCCD00972 
                 CODH_ACS 
                 bifunctional 
               
               
                   
                   
                 Dehydrogenase/Acetyl- 
                   
                   
                   
                 CODH/ACS 
               
               
                   
                   
                 CoA Synthase 
                   
                   
                   
                 enzyme, 
               
               
                   
                   
                   
                   
                   
                   
                 carbon fixation 
               
               
                   
                 6 
                 Formate Dehydrogenase 
                 1.2.1.2 
                 RCCD01275 
                 FDH_1 
                 Methyl branch 
               
               
                   
                 7 
                   
                   
                 RCCD01472 
                 FDH_2 
                 carbon fixation 
               
               
                   
                 8 
                 Formyltetrahydrofolate 
                 6.3.4.3 
                 RCCD00982 
                 FTHFS 
                 Methyl branch 
               
               
                   
                   
                 Synthase 
                   
                   
                   
                 carbon fixation 
               
               
                   
                 9 
                 Methenyltetrahydrofolate 
                 3.5.4.9 
                 RCCD00980 
                 MEC 
                 Methyl branch 
               
               
                   
                   
                 cyclohydrolase 
                   
                   
                   
                 carbon fixation 
               
               
                   
                 10 
                 Methylenetetrahydrofolate 
                 1.5.1.5 
                 RCCD00980 
                 MED 
                 Methyl branch 
               
               
                   
                   
                 dehydrogenase 
                   
                   
                   
                 carbon fixation 
               
               
                   
                 11 
                 Methylenetetrahydrofolate 
                 1.5.1.20 
                 RCCD00978 
                 MER 
                 Methyl branch 
               
               
                   
                   
                 reductase 
                   
                   
                   
                 carbon fixation 
               
               
                   
                 12 
                 Methyltransferase 
                 2.1.1.13 
                 RCCD00973 
                 MET 
                 Methyl branch 
               
               
                   
                   
                   
                   
                   
                   
                 carbon fixation 
               
               
                   
                 13 
                 Corrinoid/Iron-sulfur protein 
                 1.2.99.2 
                 RCCD00974 
                 COPL 
                 Part of 
               
               
                   
                   
                   
                   
                   
                   
                 CODH/ACS 
               
               
                   
                   
                   
                   
                   
                   
                 complex, 
               
               
                   
                   
                   
                   
                   
                   
                 Large subunit 
               
               
                   
                 14 
                 Corrinoid/Iron-sulfur protein 
                 1.2.99.2 
                 RCCD00975 
                 COPS 
                 Part of 
               
               
                   
                   
                   
                   
                   
                   
                 CODH/ACS 
               
               
                   
                   
                   
                   
                   
                   
                 complex, Small 
               
               
                   
                   
                   
                   
                   
                   
                 subunit 
               
               
                 Ethanol 
                 15 
                 Acetate Kinase 
                 2.7.2.1 
                 RCCD02720 
                 ACK 
                 Acetate 
               
               
                 and 
                   
                   
                   
                   
                   
                 production 
               
               
                 acetate 
                 16 
                 Phospho-transacetylase 
                 2.3.1.8 
                 RCCD02719 
                 PTA 
                 Acetate 
               
               
                 production 
                   
                   
                   
                   
                   
                 production 
               
               
                   
                 17 
                 Tungsten-containing 
                 1.2.7.5 
                 RCCD01679 
                 AOR_1 
                 Reduction of 
               
               
                   
                   
                 aldehyde ferredoxin 
                   
                   
                   
                 acetate to 
               
               
                   
                   
                 oxidoreductase 
                   
                   
                   
                 acetaldehyde 
               
               
                   
                 18 
                   
                 1.2.7.5 
                 RCCD01692 
                 AOR_2 
                 Reduction of 
               
               
                   
                   
                   
                   
                   
                   
                 acetate to 
               
               
                   
                   
                   
                   
                   
                   
                 acetaldehyde 
               
               
                   
                 19 
                 Alcohol Dehydrogenase 
                 1.1.1.2 
                 RCCD00257 
                 ADH_1 
                 zinc 
               
               
                   
                   
                   
                   
                   
                   
                 containing, 
               
               
                   
                   
                   
                   
                   
                   
                 NADPH 
               
               
                   
                   
                   
                   
                   
                   
                 dependent 
               
               
                   
                   
                   
                   
                   
                   
                 secondary 
               
               
                   
                   
                   
                   
                   
                   
                 ADH 
               
               
                   
                 20 
                   
                 1.1.1.1 
                 RCCD00167 
                 ADH_2 
                 two pfam 
               
               
                   
                   
                   
                   
                   
                   
                 domain: 
               
               
                   
                   
                   
                   
                   
                   
                 FeADH and 
               
               
                   
                   
                   
                   
                   
                   
                 ALDH, AdhE 
               
               
                   
                 21 
                   
                 1.1.1.1 
                 RCCD00168 
                 ADH_3 
                 two pfam 
               
               
                   
                   
                   
                   
                   
                   
                 domain: 
               
               
                   
                   
                   
                   
                   
                   
                 FeADH and 
               
               
                   
                   
                   
                   
                   
                   
                 ALDH, AdhE 
               
               
                   
                 22 
                   
                 1.1.1.1 
                 RCCD02628 
                 ADH_5 
                 one pfam 
               
               
                   
                   
                   
                   
                   
                   
                 domain: 
               
               
                   
                   
                   
                   
                   
                   
                 FeADH 
               
               
                   
                 23 
                   
                 1.1.1.1 
                 RCCD03350 
                 ADH_7 
                 one pfam 
               
               
                   
                   
                   
                   
                   
                   
                 domain: 
               
               
                   
                   
                   
                   
                   
                   
                 FeADH 
               
               
                   
                 24 
                   
                 1.—.—.— 
                 RCCD00470 
                 ADH_8 
                 short chain 
               
               
                   
                   
                   
                   
                   
                   
                 ADH, multiple 
               
               
                   
                   
                   
                   
                   
                   
                 copy 
               
               
                   
                 25 
                   
                 1.—.—.— 
                 RCCD01665 
                 ADH_9 
                 short chain 
               
               
                   
                   
                   
                   
                   
                   
                 ADH, multiple 
               
               
                   
                   
                   
                   
                   
                   
                 copy 
               
               
                   
                 26 
                   
                 1.—.—.— 
                 RCCD01767 
                 ADH_10 
                 short chain 
               
               
                   
                   
                   
                   
                   
                   
                 ADH, multiple 
               
               
                   
                   
                   
                   
                   
                   
                 copy 
               
               
                   
                 27 
                   
                 1.—.—.— 
                 RCCD02864 
                 ADH_11 
                 short chain 
               
               
                   
                   
                   
                   
                   
                   
                 ADH, multiple 
               
               
                   
                   
                   
                   
                   
                   
                 copy 
               
               
                   
                 28 
                 Aldehyde Dehydrogenase 
                 1.2.1.10 
                 RCCD02636 
                 ALDH_1 
                 Acetylating 
               
               
                   
                 29 
                   
                 1.2.1.10 
                 RCCD03356 
                 ALDH_2 
                 Acetylating 
               
               
                   
                 30 
                   
                 1.2.1.10 
                 RCCD03368 
                 ALDH_3 
                 Acetylating 
               
               
                 Hydrogenase 
                 31 
                 Hydrogenase 
                 1.12.7.2 
                 RCCD00346 
                 HYD_1 
                 Ni—Fe large 
               
               
                   
                   
                   
                   
                   
                   
                 subunit, H2 
               
               
                   
                   
                   
                   
                   
                   
                 oxidation 
               
               
                   
                 32 
                   
                 1.12.7.2 
                 RCCD00938 
                 HYD_2 
                 Ni—Fe small 
               
               
                   
                   
                   
                   
                   
                   
                 subunit, H2 
               
               
                   
                   
                   
                   
                   
                   
                 oxidation 
               
               
                   
                 33 
                   
                 1.12.7.2 
                 RCCD01283 
                 HYD_3 
                 Fe only, large 
               
               
                   
                   
                   
                   
                   
                   
                 subunit, H2 
               
               
                   
                   
                   
                   
                   
                   
                 production 
               
               
                   
                 34 
                   
                 1.12.7.2 
                 RCCD01700 
                 HYD_4 
                 Fe only, H2 
               
               
                   
                   
                   
                   
                   
                   
                 production 
               
               
                   
                 35 
                   
                 1.12.7.2 
                 RCCD02918 
                 HYD_5 
                 Fe only, H2 
               
               
                   
                   
                   
                   
                   
                   
                 production 
               
               
                   
                 36 
                   
                 1.12.7.2 
                 RCCD04233 
                 HYD_6 
                 Fe only, H2 
               
               
                   
                   
                   
                   
                   
                   
                 production 
               
               
                 Electron 
                 37 
                 Ferredoxin 
                   
                 RCCD00424 
               
               
                 carrier 
                 38 
                   
                   
                 RCCD01226 
               
               
                   
                 39 
                   
                   
                 RCCD01932 
               
               
                   
                 40 
                   
                   
                 RCCD02185 
               
               
                   
                 41 
                   
                   
                 RCCD02239 
               
               
                   
                 42 
                   
                   
                 RCCD02268 
               
               
                   
                 43 
                   
                   
                 RCCD02580 
               
               
                   
                 44 
                   
                   
                 RCCD03406 
               
               
                   
                 45 
                   
                   
                 RCCD03640 
               
               
                   
                 46 
                   
                   
                 RCCD03676 
               
               
                   
                 47 
                   
                   
                 RCCD04306 
               
               
                 Electron 
                 48 
                 Pyridine nucleotide- 
                   
                 RCCD00185 
                   
                 glutamate 
               
               
                 transfer 
                   
                 disulphide oxidoreductases 
                   
                   
                   
                 synthase small 
               
               
                   
                   
                   
                   
                   
                   
                 chain, but no 
               
               
                   
                   
                   
                   
                   
                   
                 large chain 
               
               
                   
                   
                   
                   
                   
                   
                 next to it 
               
               
                   
                 49 
                   
                   
                 RCCD01487 
                   
                 next to cooF 
               
               
                   
                   
                   
                   
                   
                   
                 and cooS, 
               
               
                   
                   
                   
                   
                   
                   
                 probably 
               
               
                   
                   
                   
                   
                   
                   
                 important for 
               
               
                   
                   
                   
                   
                   
                   
                 reduced 
               
               
                   
                   
                   
                   
                   
                   
                 pyridine 
               
               
                   
                   
                   
                   
                   
                   
                 cofactor 
               
               
                   
                   
                   
                   
                   
                   
                 generation 
               
               
                   
                 50 
                   
                   
                 RCCD00433 
                   
                 NADH 
               
               
                   
                   
                   
                   
                   
                   
                 dehydrogenase, 
               
               
                   
                   
                   
                   
                   
                   
                 not part of 
               
               
                   
                   
                   
                   
                   
                   
                 an operon 
               
               
                   
                 51 
                 Membrane-associated 
                   
                 RCCD01488 
                 cooF 
                 Between gene 
               
               
                   
                   
                 electron transfer FeS 
                   
                   
                   
                 number 3 and 
               
               
                   
                   
                 protein, cooF 
                   
                   
                   
                 gene number 
               
               
                   
                   
                   
                   
                   
                   
                 49 
               
               
                   
               
             
          
         
       
     
     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. 
     
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                   Clostridium carboxidivorans  genes used in metabolic engineering. 
               
             
          
           
               
                   
                 Gene 
                   
                 EC 
                   
                   
                   
               
               
                 Pathway 
                 ID 
                 Gene Name 
                 number 
                 ORF ID 
                 Copy ID 
                 Description 
               
               
                   
               
             
          
           
               
                 Wood- 
                 1 
                 Carbon Monoxide 
                 1.2.2.4 
                 RCCB04039 
                 CODH_1 
                 CO oxidation 
               
               
                 Ljungdahl 
                 2 
                 Dehydrogenase 
                   
                 RCCB01154 
                 CODH_2 
                 CO oxidation 
               
               
                   
                 3 
                   
                   
                 RCCB02478 
                 CODH_3 
                 CO oxidation 
               
               
                   
                 4 
                   
                   
                 RCCB03963 
                 CODH_4 
                 CO oxidation 
               
               
                   
                 5 
                   
                   
                 RCCB04038 
                 CODH_5 
                 CO oxidation 
               
               
                   
                 6 
                 Carbon Monoxide 
                 1.2.99.2 
                 RCCB04293 
                 CODH_ACS 
                 bifunctional 
               
               
                   
                   
                 Dehydrogenase/Acetyl- 
                   
                   
                   
                 CODH/ACS 
               
               
                   
                   
                 CoA Synthase 
                   
                   
                   
                 enzyme, carbon 
               
               
                   
                   
                   
                   
                   
                   
                 fixation 
               
               
                   
                 7 
                 Formate Dehydrogenase 
                 1.2.1.2 
                 RCCB05406 
                 FDH_1 
                 Methyl branch 
               
               
                   
                 8 
                   
                   
                 RCCB01346 
                 FDH_2 
                 carbon fixation 
               
               
                   
                 9 
                 Formyltetrahydrofolate 
                 6.3.4.3 
                 RCCB04040 
                 FTHFS 
                 Methyl branch 
               
               
                   
                   
                 Synthase 
                   
                   
                   
                 carbon fixation 
               
               
                   
                 10 
                 Methenyltetrahydrofolate 
                 3.5.4.9 
                 RCCB04042 
                 MEC 
                 Methyl branch 
               
               
                   
                   
                 cyclohydrolase 
                   
                   
                   
                 carbon fixation 
               
               
                   
                 11 
                 Methylenetetrahydrofolate 
                 1.5.1.5 
                 RCCB04042 
                 MED 
                 Methyl branch 
               
               
                   
                   
                 dehydrogenase 
                   
                   
                   
                 carbon fixation 
               
               
                   
                 12 
                 Methylenetetrahydrofolate 
                 1.5.1.20 
                 RCCB04044 
                 MER 
                 Methyl branch 
               
               
                   
                   
                 reductase 
                   
                   
                   
                 carbon fixation 
               
               
                   
                 13 
                 Methyltransferase 
                 2.1.1.13 
                 RCCB04294 
                 MET 
                 Methyl branch 
               
               
                   
                   
                   
                   
                   
                   
                 carbon fixation 
               
               
                   
                 14 
                 Corrinoid/Iron-sulfur 
                 1.2.99.2 
                 RCCB04049 
                 COPL 
                 Part of CODH/ACS 
               
               
                   
                   
                 protein 
                   
                   
                   
                 complex, Large 
               
               
                   
                   
                   
                   
                   
                   
                 subunit 
               
               
                   
                 15 
                 Corrinoid/Iron-sulfur 
                 1.2.99.2 
                 RCCB04047 
                 COPS 
                 Part of CODH/ACS 
               
               
                   
                   
                 protein 
                   
                   
                   
                 complex, Small 
               
               
                   
                   
                   
                   
                   
                   
                 subunit 
               
               
                 Ethanol 
                 16 
                 Acetate Kinase 
                 2.7.2.1 
                 RCCB05249 
                 ACK 
                 Acetate production 
               
               
                 and 
                 17 
                 Phospho-transacetylase 
                 2.3.1.8 
                 RCCB02481 
                 PTA 
                 Acetate production 
               
               
                 acetate 
                 18 
                 Tungsten-containing 
                 1.2.7.5 
                 RCCB00063 
                 AOR_1 
                 Reduction of 
               
               
                 production 
                   
                 aldehyde ferredoxin 
                   
                   
                   
                 acetate to 
               
               
                   
                   
                 oxidoreductase 
                   
                   
                   
                 acetaldehyde 
               
               
                   
                 19 
                 Alcohol Dehydrogenase 
                 1.1.1.2 
                 RCCB03584 
                 ADH_1 
                 zinc-ADH 
               
               
                   
                 20 
                   
                 1.1.1.1 
                 RCCB03870 
                 ADH_2 
                 two pfam domain: 
               
               
                   
                   
                   
                   
                   
                   
                 FeADH and ALDH, 
               
               
                   
                   
                   
                   
                   
                   
                 AdhE 
               
               
                   
                 21 
                   
                 1.1.1.1 
                 RCCB05675 
                 ADH_3 
                 truncated, AdhE 
               
               
                   
                 22 
                   
                 1.1.1.1 
                 RCCB00958 
                 ADH_5 
                 one pfam domain: 
               
               
                   
                   
                   
                   
                   
                   
                 FeADH 
               
               
                   
                 23 
                   
                 1.1.1.1 
                 RCCB04489 
                 ADH_6 
                 one pfam domain: 
               
               
                   
                   
                   
                   
                   
                   
                 FeADH 
               
               
                   
                 24 
                   
                 1.1.1.1 
                 RCCB04503 
                 ADH_7 
                 one pfam domain: 
               
               
                   
                   
                   
                   
                   
                   
                 FeADH 
               
               
                   
                 25 
                   
                 1.—.—.— 
                 RCCB02465 
                 ADH_9 
                 short chain ADH, 
               
               
                   
                   
                   
                   
                   
                   
                 multiple copy 
               
               
                   
                 26 
                   
                 1.—.—.— 
                 RCCB05551 
                 ADH_10 
                 short chain ADH, 
               
               
                   
                   
                   
                   
                   
                   
                 multiple copy 
               
               
                   
                 27 
                 Aldehyde 
                 1.2.1.10 
                 RCCB02403 
                 ALDH_1 
                 Acetylating 
               
               
                   
                 28 
                 Dehydrogenase 
                 1.2.1.10 
                 RCCB02561 
                 ALDH_2 
                 Acetylating 
               
               
                   
                 29 
                   
                 1.2.1.10 
                 RCCB04031 
                 ALDH_3 
                 Acetylating 
               
               
                 Hydrogenase 
                 30 
                 Hydrogenase 
                 1.12.7.2 
                 RCCB02249 
                 HYD_1 
                 Ni—Fe large subunit, 
               
               
                   
                   
                   
                   
                   
                   
                 H2 oxidation 
               
               
                   
                 31 
                   
                 1.12.7.2 
                 RCCB01319 
                 HYD_2 
                 Fe only, H2 
               
               
                   
                   
                   
                   
                   
                   
                 production 
               
               
                   
                 32 
                   
                 1.12.7.2 
                 RCCB01405 
                 HYD_3 
                 Fe only, H2 
               
               
                   
                   
                   
                   
                   
                   
                 production 
               
               
                   
                 33 
                   
                 1.12.7.2 
                 RCCB01516 
                 HYD_4 
                 Fe only, large 
               
               
                   
                   
                   
                   
                   
                   
                 subunit, H2 
               
               
                   
                   
                   
                   
                   
                   
                 production 
               
               
                   
                 34 
                   
                 1.12.7.2 
                 RCCB03483 
                 HYD_5 
                 Fe only, H2 
               
               
                   
                   
                   
                   
                   
                   
                 production 
               
               
                   
                 35 
                   
                 1.12.7.2 
                 RCCB05411 
                 HYD_6 
                 Fe only, large 
               
               
                   
                   
                   
                   
                   
                   
                 subunit, H2 
               
               
                   
                   
                   
                   
                   
                   
                 production 
               
               
                 Electron 
                 36 
                 Ferredoxin 
                   
                 RCCB00234 
               
               
                 carrier 
                 37 
                   
                   
                 RCCB00345 
               
               
                   
                 38 
                   
                   
                 RCCB01260 
               
               
                   
                 39 
                   
                   
                 RCCB01334 
               
               
                   
                 40 
                   
                   
                 RCCB01775 
               
               
                   
                 41 
                   
                   
                 RCCB01960 
               
               
                   
                 42 
                   
                   
                 RCCB01972 
               
               
                   
                 43 
                   
                   
                 RCCB02618 
               
               
                   
                 44 
                   
                   
                 RCCB02638 
               
               
                   
                 45 
                   
                   
                 RCCB02836 
               
               
                   
                 46 
                   
                   
                 RCCB02853 
               
               
                   
                 47 
                   
                   
                 RCCB03023 
               
               
                   
                 48 
                   
                   
                 RCCB03191 
               
               
                   
                 49 
                   
                   
                 RCCB03278 
               
               
                   
                 50 
                   
                   
                 RCCB03452 
               
               
                   
                 51 
                   
                   
                 RCCB03596 
               
               
                   
                 52 
                   
                   
                 RCCB03762 
               
               
                   
                 53 
                   
                   
                 RCCB03972 
               
               
                   
                 54 
                   
                   
                 RCCB04165 
               
               
                   
                 55 
                   
                   
                 RCCB04383 
               
               
                   
                 56 
                   
                   
                 RCCB04571 
               
               
                   
                 57 
                   
                   
                 RCCB04585 
               
               
                   
                 58 
                   
                   
                 RCCB05780 
               
               
                   
                 59 
                   
                   
                 RCCB05975 
               
               
                   
                 60 
                   
                   
                 RCCB06304 
               
               
                   
                 61 
                   
                   
                 RCCB06305 
               
               
                 Electron 
                 62 
                 Pyridine nucleotide- 
                   
                 RCCB00442 
                   
                 NADH 
               
               
                 transfer 
                   
                 disulphide 
                   
                   
                   
                 dehydrogenase, 
               
               
                   
                   
                 oxidoreductases 
                   
                   
                   
                 not part of an 
               
               
                   
                   
                   
                   
                   
                   
                 operon 
               
               
                   
                 63 
                   
                   
                 RCCB01674 
                   
                 NADH 
               
               
                   
                   
                   
                   
                   
                   
                 dehydrogenase, 
               
               
                   
                   
                   
                   
                   
                   
                 not part of an 
               
               
                   
                   
                   
                   
                   
                   
                 operon 
               
               
                   
                 64 
                   
                   
                 RCCB03510 
                   
                 next to cooF and 
               
               
                   
                   
                   
                   
                   
                   
                 cooS, probably 
               
               
                   
                   
                   
                   
                   
                   
                 important for 
               
               
                   
                   
                   
                   
                   
                   
                 reduced pyridine 
               
               
                   
                   
                   
                   
                   
                   
                 cofactor generation 
               
               
                   
                 65 
                   
                   
                 RCCB00586 
                   
                 NADH 
               
               
                   
                   
                   
                   
                   
                   
                 dehydrogenase, 
               
               
                   
                   
                   
                   
                   
                   
                 not part of an 
               
               
                   
                   
                   
                   
                   
                   
                 operon 
               
               
                   
                 66 
                   
                   
                 RCCB04795 
                   
                 NADH:ferredoxin 
               
               
                   
                   
                   
                   
                   
                   
                 oxidoreductase, not 
               
               
                   
                   
                   
                   
                   
                   
                 part of an operon 
               
               
                   
                 67 
                 Membrane-associated 
                   
                 RCCB03509 
                 cooF 
                 Between gene 
               
               
                   
                   
                 electron transfer FeS 
                   
                   
                   
                 number 2 and gene 
               
               
                   
                   
                 protein, cooF 
                   
                   
                   
                 number 64 
               
               
                   
               
             
          
         
       
     
     In addition to ethanol synthesis,  C. carboxydivorans  produces butanol, butyrate, hexanol and hexanoate. The genes coding for the enzymes in the butanol/butyrate synthetic pathway were identified and located within the genome of  C. carboxydivorans  as shown in Table 4. 
     
       
         
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                   Clostridium carboxidivorans  butanol production genes 
               
             
          
           
               
                   
                 Gene 
                   
                 EC 
                   
                   
                   
               
               
                 Pathway 
                 ID 
                 Gene Name 
                 number 
                 ORF ID 
                 Copy ID 
                 Description 
               
               
                   
               
               
                 Butanol 
                 70 
                 Acetyl-CoA 
                 2.3.1.9 
                 RCCB05104 
                   
                   
               
               
                 production 
                 71 
                 acetyltransferase 
                   
                 RCCB06240 
               
               
                   
                 72 
                   
                   
                 RCCB04397 
               
               
                   
                 73 
                 3-hydroxybutyryl- 
                 1.1.1.157 
                 RCCB05103 
               
               
                   
                 74 
                 CoA dehydrogenase 
                   
                 RCCB03354 
               
               
                   
                 75 
                 3-hydroxyacyl-CoA 
                 1.1.1.35 
                 RCCB01361 
               
               
                   
                   
                 dehydrogenase 
               
               
                   
                 76 
                 3-hydroxybutyryl- 
                 4.2.1.55 
                 RCCB03353 
               
               
                   
                 77 
                 CoA dehydratase 
                   
                 RCCB05102 
               
               
                   
                 78 
                 Butyryl-CoA 
                 1.3.99.2 
                 RCCB01169 
               
               
                   
                 79 
                 dehydrogenase 
                   
                 RCCB05105 
               
               
                   
                 80 
                   
                   
                 RCCB03214 
               
               
                   
                 81 
                   
                   
                 RCCB01307 
               
               
                   
                 82 
                   
                   
                 RCCB00309 
               
               
                   
                 83 
                   
                   
                 RCCB04399 
               
               
                   
                 84 
                 Phosphate 
                 2.3.1.19 
                 RCCB03974 
               
               
                   
                 85 
                 butyryltransferase 
                   
                 RCCB01389 
               
               
                   
                 86 
                 Butyrate kinase 
                 2.7.2.7 
                 RCCB01073 
               
               
                   
                 87 
                   
                   
                 RCCB00672 
               
               
                   
                 88 
                   
                   
                 RCCB03973 
               
               
                   
                 89 
                 Butanal/butanol 
                 1.2.1.57 
                 RCCB05526 
               
               
                   
                 90 
                 dehydrogenase 
                   
                 RCCB05675 
               
               
                   
                 91 
                   
                   
                 RCCB03870 
               
               
                   
               
             
          
         
       
     
     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. 
       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). 
     Six hydrogenase (EC 1.12.7.2) ORF sequences were identified in the genome of each of the acetogenic  Clostridium  species. 
     Twelve ferredoxin biosynthesis genes (Gene ID 40-51, Table 1) 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.    
     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 potentially 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 potentially key gene RCCD01847, is part of the cooF/cooS operon shown in  FIG. 3 . 
     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 ). 
     The genes encoding the NADPH-dependent secondary alcohol dehydrogenase (Gene ID 21, Table 1; Gene ID 19, Table 2) were sequenced in  C. ragsdalei  and  C. ljungdahlii.  Alignment 400 of the NADPH-dependent secondary alcohol dehydrogenase proteins of the acetogenic Clostridiae with the experimentally confirmed NADPH-dependent secondary alcohol dehydrogenase from  Thermoanaerobacter ethanolicus  are shown in  FIG. 4 . The  Thermoanaerobacter ethanolicus  NADPH-dependent alcohol dehydrogenase has been sequenced and purified. The anaerobic micro-organism is known to produce NADPH-dependent alcohol dehydrogenase activity having the amino acid sequence shown in  FIG. 4 . Coloration in alignment 400 indicates the degree of identity and similarity at each amino acid position in the three organisms. Asterisks indicate amino acids that are identical at the same position in all three organisms. Single dots above the amino acids indicate general similarity among the three proteins. Two dots indicate very similar amino acids among the three organisms. No dots or asterisks above the amino acids indicates no similarity. A high degree of gene conservation is observed for the NADPH-dependent secondary alcohol dehydrogenase enzyme in  C. ragsdalei  and  C. ljungdahlii.  Furthermore, in all three micro-organisms, the NADPH-dependent alcohol dehydrogenase exhibits a high degree of homology. 
     Key genes to modulate production of aliphatic C 2 -C 6  alcohols and acids in  C. ragsdahlei  include: 
     SEQ ID NO 1 (ORF #RCCC02026, Table 1) the gene sequence for carbon monoxide dehydrogenase, cooS; 
     SEQ ID NO 2 (ORF #RCCC02027, Table 1), the gene sequence for electron transfer FeS protein cooF; 
     SEQ ID NO 3 (ORF #RCCC02028, Table 1), the gene sequence for the NADH dependent ferredoxin oxidoreductase (FOR); 
     SEQ ID NO 4 (ORF #RCCC01718, Gene ID 17, Table 1,  FIG. 2 ,  FIG. 5 ), the gene sequence for a phospho-transacetylase enzyme involved in acetate production; 
     SEQ ID NO 5 (ORF #RCCC01717, Gene ID 16, Table 1,  FIG. 2 ,  FIG. 5 ), the gene sequence for acetate kinase, an enzyme involved in acetate production; 
     SEQ ID NO 6 (ORF #RCCC02715, Gene ID 21, Table 1,  FIG. 2 ,  FIG. 5 ), the gene sequence for NADPH dependent secondary alcohol dehydrogenase; 
     SEQ ID NO 7 (ORF #RCCC01356, Gene ID 22, Table 1), the gene sequence for an adhE-type alcohol dehydrogenase; 
     SEQ ID NO 8 (ORF #RCCC01357, Gene ID 23, Table 1), the gene sequence for an adhE-type alcohol dehydrogenase; 
     SEQ ID NO 9 (ORF #RCCC01358, Gene ID 24, Table 1), the gene sequence for an adhE-type alcohol dehydrogenase, truncated; 
     SEQ ID NO 10 (ORF #RCCC03300, Gene ID 25, Table 1), the gene sequence for an iron-containing alcohol dehydrogenase; 
     SEQ ID NO 11 (ORF #RCCC03712, Gene ID 26, Table 1), the gene sequence for an iron-containing alcohol dehydrogenase; 
     SEQ ID NO 12 (ORF #RCCC04095, Gene ID 27, Table 1), the gene sequence for an iron-containing alcohol dehydrogenase; 
     SEQ ID NO 13 (ORF #RCCC0004, Gene ID 28, Table 1), the gene sequence for a short-chain alcohol dehydrogenase; 
     SEQ ID NO 14 (ORF #RCCC1567, Gene ID 29, Table 1), the gene sequence for a short-chain alcohol dehydrogenase; 
     SEQ ID NO 15 (ORF #RCCC2765, Gene ID 30, Table 1), the gene sequence for a short-chain alcohol dehydrogenase; 
     SEQ ID NO 16 (ORF RCCC2240, Gene ID 31, Table 1), the gene sequence for a short-chain alcohol dehydrogenase; 
     SEQ ID NO 17 (ORF #RCCC00020, Gene ID 18, Table 1), the gene sequence for an aldehyde ferredoxin oxidoreductase; 
     SEQ ID NO 18 (ORF #RCCC0030, Gene ID 19, Table 1), the gene sequence for an aldehyde ferredoxin oxidoreductase; 
     SEQ ID NO 19 (ORF #RCCC01183, Gene ID 20, Table 1), the gene sequence for an aldehyde ferredoxin ixidoreductase. 
     Key genes to modulate production of ethanol and acetate in  C. ljungdahlii  include: 
     SEQ ID NO 20 (ORF #RCCD00257, Gene ID 19, Table 2) the gene sequence for NADPH dependent secondary alcohol dehydrogenase; 
     SEQ ID NO 21 (ORF #RCCD00167, Gene ID 20, Table 2) the gene sequence for an adhE-type alcohol dehydrogenase; 
     SEQ ID NO 22 (ORF #RCCD00168, Gene ID 21, Table 2) the gene sequence for an adhE-type alcohol dehydrogenase; 
     SEQ ID NO 23 (ORF #RCCD02628, Gene ID 22, Table 2) the gene sequence for an adhE-type alcohol dehydrogenase; 
     SEQ ID NO 24 (ORF #RCCD03350, Gene ID 23, Table 2) the gene sequence for an adhE-type alcohol dehydrogenase; 
     SEQ ID NO 25 (ORF #RCCD00470, Gene ID 24, Table 2) the gene sequence for a short-chain alcohol dehydrogenase; 
     SEQ ID NO 26 (ORF #RCCD01665, Gene ID 25, Table 2) the gene sequence for a short-chain alcohol dehydrogenase; 
     SEQ ID NO 27 (ORF #RCCD01767, Gene ID 26, Table 2) the gene sequence for a short-chain alcohol dehydrogenase; 
     SEQ ID NO 28 (ORF #RCCD02864, Gene ID 27, Table 2) the gene sequence for a short-chain alcohol dehydrogenase; 
     SEQ ID NO 29 (ORF #RCCD01679, Gene ID 17, Table 2) the gene sequence for an aldehyde ferredoxin oxidoreductase; and 
     SEQ ID NO 30 (ORF #RCCD01692, Gene ID 18, Table 2) the gene sequence for an aldehyde ferredoxin oxidoreductase. 
     Key genes to modulate production of butanol and butyrate in  C. carboxydivorans  include: 
     SEQ ID NO 31 (ORF #RCCB03972, Gene ID 84, Table 4,  FIG. 8 ), the gene sequence for phosphate butyryltransferase; and 
     SEQ ID NO 32 (ORF #RCCB03973, Gene ID 88, Table 4,  FIG. 8 ), the gene sequence for butyrate kinase. 
     Using the detailed genomic information in Tables 1 through 4 and in the Sequence Listing, 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 pathways have been modulated. In one embodiment, gene expression of key alcohol producing enzymes is increased (overexpression) 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 NADPH-dependent secondary alcohol dehydrogenase (21,  FIG. 2  and Table 1) and aldehyde ferredoxin oxidoreductase are duplicated within the genome of the modified organism. Alternatively, ethanol production can be increased by conversion of acetyl-CoA 102 first to acetaldehyde 604, then to ethanol 216 by overexpressing one or more of the other alcohol dehydrogenases present in the Clostridia micro-organisms&#39; genome such as adhE-type alcohol dehydrogenase, iron-containing alcohol dehydrogenase, and short-chain alcohol dehydrogenases (21-31, Table 1; Gene ID 20-30, Table 2). 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. 
     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. 
     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. 
     Example 1 
     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 oxidized nucleotide cofactors NAD +  and NADP +.  The increased levels of reduced nucleotide cofactors (NADH and NADPH) then stimulate generation of intermediate compounds in Wood-Ljungdahl pathway 100. 
     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, Wis.) or pTOPO (Invitrogen, Carlsbad, Calif.). 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. 
     Example 2 
     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 102 to acetyl-phosphate 218, and acetate kinase (Gene ID16, 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. 5 . 
     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. 5  by X 504 and X 502, respectively. 
     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. 
     Example 3 
     Secondary alcohol dehydrogenases are known to convert ketones to secondary alcohols. In vivo, the NADPH-dependent secondary alcohol dehydrogenase enzyme (Gene ID 21 in  FIG. 5 , Table 1, Gene ID 19, Table2) also converts the Coenzyme A (CoA) form of a carbon moiety, such as acetyl-CoA 102 or butyrl-CoA 806 ( FIG. 8 ), 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. 6 , 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 secondary alcohol dehydrogenase 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. 
     In one embodiment, secondary alcohol dehydrogenase 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 secondary alcohol dehydrogenase. Confirmation of gene placement followed by gene expression studies of the recombinant strain are performed and compared to the parent strain. 
     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. 
     Example 4 
     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+P i  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. 
     Acetogenic Clostridia including,  C. ragsdahlei, C. ljungdahlii  and  C. carboxydivorans  contain the gene encoding the aldehyde ferredoxin oxidoreductase (Gene ID 18, 19, 20, Table 1; 17, 18, Table 2; and 18, Table 3) that converts acetate 214 to acetaldehyde. In one embodiment, the acetate-to-acetaldehyde conversion rate is increased in  C. ragsdahlei,  for example, as shown in  FIG. 7 . An aldehyde ferredoxin oxidoreductase (AOR) is duplicated in the genomes of  C. ragsdahlei  using methods described in several examples above. The gene is amplified and cloned into integration vectors containing acetogenic Clostridial DNA that allows insertion at a neutral site in the chromosome. Gene replacement strains are confirmed through standard molecular biological methods and acetate conversion rates will be determined using parent strains as controls. In one embodiment, acetate conversion is increased by between 10 and 20%, as indicated by vertical arrow 704. 
     In some cases, it is desirable to generate a double mutant through duplication of two genes to generate a strain that produces more ethanol through increased secondary alcohol dehydrogenase activity (21 Table 1; 19, Table 2) and also has improved acetate conversion capabilities. One embodiment of the invention, shown in  FIG. 7 , is dual mutant strain 700 of  C. ragsdahlei.  This modified organism has elevated AOR activity, shown by vertical arrow 704 due to duplicated aldehyde ferredoxin oxidoreductase. The modified organism also has elevated NADPH dependent secondary alcohol dehydrogenase (21, Table 1 and  FIG. 7 ) activity due to duplicated NADPH-dependent secondary alcohol dehydrogenase, indicated in  FIG. 7  by vertical arrow 706. 
     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 AOR and NADPH-dependent secondary alcohol dehydrogenase 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 micro-organism. 
     Example 5 
     Certain acetogenic Clostridia,  C. carboxydivorans  for example, produce significant amounts of C 4 -C 6  alcohols and their corresponding acids in addition to ethanol and acetate. In some cases, a mix of alcohols is desirable for industrial production. As shown in  FIG. 8 , strategy 800, to be employed for increasing the production of C 4 -C 6  alcohols in acetogenic Clostridia, is to block the strain&#39;s ability to produce butyrate 802 after the production of crotonyl-CoA 804. Similar to the phosphotransacetylase and acetate kinase operons identified in many acetogenic Clostridia, the genes for the conversion of butyryl-CoA 806 to butyrate 802 exist as one operon, comprising both phosphate butyryltransferase (Gene ID 84, 85, Table 4) and butyrate kinase (Gene ID 86, 87, 88, Table 4). The operon comprising the two butyrate production genes will be deleted from  C. carboxydivorans.  The desired recombinant strain will lack, or be attenuated in the transferase and kinase activities, as indicated by X 808 and X 810, respectively in  FIG. 8 . 
     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.

Technology Classification (CPC): 8