Making C4+ products in bacteria

Abstract

Methods of making C4+ hydrocarbon feedstocks using anaerobic microbes are described.

Claims

1. A method for the bioproduction of a C4+ compound, said method comprising: a. anaerobically growing a bacteria in a culture medium comprising a carbon source; b. conversion of said carbon source to a C4+ compound; and c. purification of said C4+ compound, said bacteria being a genetically engineered bacteria comprising an overexpressed ferredoxin-NAD(P)+ reductase (FNR) capable of catalyzing reduction of either NADP+ or NAD+ or both, said bacteria able to anaerobically produce more C4-8 organic acids or alcohols than a similar bacteria lacking said overexpressed FNR.

2. The method of claim 1, wherein said C4+ compound is butyrate, butanol, valeric acid, pentanol, hexanoate, hexanol, heptanoate, heptanol, octanoate, octanol, or a derivative thereof.

3. The method of claim 1, wherein said C4+ compound is butyrate.

4. The method of claim 1, wherein said C4+ compound is butanol.

5. The method of claim 1, wherein said growing step is anaerobic.

6. The method of claim 1, wherein said growing step is 100% anaerobic.

7. The method of claim 1, wherein said growing step is <100% anaerobic.

8. The method of claim 1, wherein said FNR is a heterologous FNR.

9. The method of claim 1, said bacteria further comprising a mutation such that said bacteria cannot make lactate.

10. The method of claim 1, wherein said bacteria is an acetogenic bacteria.

11. The method of claim 1, wherein said bacteria is a Clostridium, Clostridium acetobutylicum, Clostridium acetobutylicum M5, Clostridium thermocellum, Clostridium ljungdahlii, Clostridium thermoautotrophicum, or Clostridium tyrobutyricum.

12. The method of claim 1, wherein said bacteria is a Butyrobacterium, Moorella thermoacetica, Sporomusa, Thermacetogenium phaeum, Acetogenium kivui, Acetobacterium woodii, or Eubacterium.

13. The method of claim 10, said bacteria further comprising a mutation such that said bacteria cannot make acetone.

14. The method of claim 10, said bacteria further comprising a mutation such that said bacteria cannot make lactate.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1. Metabolic pathways in C. acetobutylicum. Ferredoxin NAD(P) reductase (FNR) catalyzed reaction of utilizing redox while reducing NAD(P) to NAD(P)H is shown in the box. pfor: pyruvate ferredoxin oxidoreductase; ack: acetate kinase; pta: phosphate acetyltransferase; adhE: aldehyde alcohol dehydrogenase; bdhAB: butanol dehydrogenase; edh: ethanol dehydrogenase; thl: acetoacetyl-CoA thiolase; hbd: 3-hydroxybutyryl-CoA dehydrogenase; adc: acetoacetate decarboxylase; ctfAB: butyrate-acetoacetate CoA-transferase; crt: 3-hydroxybutyryl-CoA dehydratase; bcd: butyryl-CoA dehydrogenase; ptb: phosphotransbutyrylase; buk: butyrate kinase.

(2) FIG. 2A SEQ ID NO. 1 Amino acid sequence of native Ferredoxin-NAD(P) reductase (FNR) of Chlorobium tepidum TLS (GenBank Accession Q8KCB2), and FIG. 2B SEQ ID NO. 2. Optimized nucleotide sequence of FNR for expression in Clostridium acetobutylicum ATCC 824. FIG. 2C SEQ ID NO. 3. Synthetic ribosome binding site.

(3) FIG. 3. Plasmid Maps: Schematic diagram showing the construction of pSOS94-FNR. The 1.1 Kb FNR (Ferredoxin-NAD(P) reductase) of Chlorobium tepidum TLS was codon optimized for expression in Clostridium acetobutylicum, synthesized with ribosome binding site (rbs), and cloned in pSOS94 using BamHI and KasI sites. The newly constructed 6.1 Kb pSOS94-FNR expresses the FNR under constitutive PTB promoter. Abbreviations: PTB, phosphotransbutyrylase promoter; ctfA, acetoacetyl-CoA:acetate/butyrate:CoA transferase subunit A; ctfB, acetoacetyl-CoA:acetate/butyrate:CoA transferase subunit B; adc, acetoacetate decarboxylase; FNR, codon optimized ferredoxin-NADP reductase gene from C. tepidum; bla, beta-lactamase for ampicillin resistance; repL, replication protein; mlsR, macrolide-lincosamide-streptogramin B resistance protein. Restriction enzyme sites: BamHI, KasI, NarI, SalI.

(4) FIG. 4. Plasmid Maps: Schematic diagram showing the construction of pJIR750-FNR. The 1.4 Kb fragment containing PTB promoter, ribosome binding site and codon optimized FNR (Ferredoxin-NADP reductase) of Chlorobium tepidum TLS was excised from pSOS94-FNR using restriction enzyme SalI, and ligated to SalI digested 6.6 Kb pJIR750. The newly constructed 7.9 Kb pJIR750-FNR expresses the FNR under constitutive PTB promoter. Abbreviations: PTB, phosphotransbutyrylase promoter; FNR, codon optimized ferredoxin-NADP reductase gene from C. tepidum; bla, beta-lactamase for ampicillin resistance; repL, replication protein; mlsR, macrolide-lincosamide-streptogramin B resistance protein; lacZ, beta-galactosidase alpha-peptide; ori, C. perfringens pIP404 replication origin; rep, replication enzyme; catP, chloramphenicol acetyltransferase; oriEC, replication region. Restriction enzyme sites: BamHI, KasI, NarI, SalI, EcoRI, SacI, KpnI, XbaI, PstI, SphI, HindIII.

(5) FIG. 5. C. acetobutylicum pathways and pSOL1 genes. Metabolic pathways in C. acetobutylicum and associated calculated in vivo fluxes. Selected enzymes are shown in bold and associated intracellular fluxes are shown in italics. Enzymes are abbreviated as follows: hydrogenase (HYDA); phosphotransacetylase (PTA); acetate kinase (AK); thiolase (THL); -hydroxybutyryl dehydrogenase (BHBD); crotonase (CRO); butyryl-CoA hydrogenase (BCD); CoA Transferase (CoAT); acetoacetate decarboxylase (AADC); butyrate kinase (BK); phosphotransbutyrylase (PTB); alcohol/aldehyde dehydrogenase (AAD). Note: AAD is believed to be the primary enzyme for butanol and ethanol formation but additional genes exist that code for alcohol forming enzymes (adhe2, bdhA, bdhB, CAC3292, CAP0059) The pathways whose genes reside on the pSOL1 megaplasmid and are absent in M5 are shown as dotted lines. The boxed pathway shows the ATP generation and NADH production occurring during metabolism.

DETAILED DESCRIPTION

(6) The disclosure relates to bacteria for making C4+ organic acids, alcohols or derivatives thereof, as well as to methods of making C4+ organic acids, alcohols or derivatives therefrom by culturing the engineered bacteria described herein with a source of carbon, forming C4+ organic acids, alcohols or derivatives, harvesting said C4+ products. The products can be used as is, or converted to other desirable compounds such as alkanes, alkenes, alcohols, esters, acids, amides, and the like.

(7) Preferred compounds made herein include the saturated C4-C8 acids (or esters thereof):

(8) TABLE-US-00004 Butyric acid Butanoic acid CH.sub.3(CH.sub.2).sub.2COOH C4:0 Valeric acid Pentanoic acid CH.sub.3(CH.sub.2).sub.3COOH C5:0 Caproic acid Hexanoic acid CH.sub.3(CH.sub.2).sub.4COOH C6:0 Enanthic acid Heptanoic acid CH.sub.3(CH.sub.2).sub.5COOH C7:0 Caprylic acid Octanoic acid CH.sub.3(CH.sub.2).sub.6COOH C8:0 Pelargonic acid Nonanoic acid CH.sub.3(CH.sub.2).sub.7COOH C9:0

(9) Other preferred products include the alcohols, butanol, pentanol, hexanol, heptanol and octanol.

(10) Preferably, the above bacteria also have reduced fermentation pathways leading to competing products, such as acetate, lactate, ethanol and/or formate. Many such mutants are already available in the art and can be used as host cells, or the vectors can be used to introduce same.

(11) Acetogens are a useful starting host, as they may contain one or more of the required enzymes (e.g., certain bacteria contain an enzyme for reaction 6), and be suitable for making C4-8 or C4-10 products. Most acetogens use the Wood-Ljungdahl pathway. The Wood-Ljungdahl pathway is a set of biochemical reactions used by some bacteria and archaea. It is also known as the reductive acetyl-CoA pathway, and enables certain organisms to use hydrogen as an electron donor and carbon dioxide as an electron acceptor as well as a building block for biosynthesis. In this pathway carbon dioxide is reduced to carbon monoxide, which is then converted to acetyl coenzyme A. Two enzymes participate, CO Dehydrogenase and acetyl-CoA synthase. The former catalyzes the reduction of the CO.sub.2 and the latter combines the resulting CO with a methyl group to give acetyl-CoA. Unlike the Reverse Krebs cycle and the Calvin cycle, this process is not cyclic.

(12) Many acetogens are thought to be strict anaerobes, thus it may be preferred to perform some of the needed engineering in a more easily grown bacteria, such as E. coli, or other commonly engineering microbe. However, acetogens are also present in aerated soils and colonize habitats with fluctuating redox conditions (e.g., the rhizosphere of sea grass), suggesting that less strict isolates are obtainable, as confirmed by Mullin's work. The use of anaerobes that are less strict may be preferred as maintaining 100% anaerobic conditions is difficult and costly.

(13) Other acetogens include Clostridium autoethanogenum, Eurobacterium limosum, Clostridium carboxidivorans P7, Peptostreptococcus products, and Butyribacterium methylotrophicum, Clostridium ljungdahlii and Acetobacterium woodii.

(14) Still other bacteria that could be useful hosts include Clostridium, Butyrobacterium, Moorella thermoacetica, Sporomusa, Thermacetogenium phaeum, Clostridium thermocellum, Acetogenium kivui, Acetobacterium woodii, Butyribacterium methylotrophicum, Clostridium ljungdahlii, Clostridium thermoautotrophicum, Clostridium tyrobutyricum, or Eubacterium limosum, or any other organism that uses ferredoxin as a major means of electron transfer factor.

(15) In more detail, the invention includes one or more of the following embodiments in any combination thereof: A genetically engineered bacteria, comprising an overexpressed ferredoxin-NAD(P)+ reductase (FNR) capable of catalyzing reduction of either NADP+ or NAD+ or both, said bacteria able to anaerobically produce more C4-8 organic acids or alcohols than a similar bacteria lacking said overexpressed FNR. A genetically engineered acetogenic bacteria, comprising an overexpressed heterologous ferredoxin-NAD(P)+ reductase (FNR) capable of efficiently catalyzing reduction of both NADP+ and NAD+, said bacteria able to anaerobically produce more C4-C8 organic acids or alcohols than a similar bacteria lacking said overexpressed heterologous FNR. A genetically engineered Clostridial bacteria, comprising an overexpressed heterologous ferredoxin-NAD(P)+ reductase (FNR) capable of efficiently catalyzing reduction of both NADP+ and NAD+, said bacteria able to anaerobically produce more C4-C8 organic acids or alcohols than a similar bacteria lacking said overexpressed heterologous FNR. A bacteria as described herein, wherein said FNR is a heterologous FNR. A bacteria as described herein, further comprising a mutation such that said bacteria cannot make acetone, lactate, formate or combinations thereof A bacteria as described herein, wherein said bacteria is an acetogenic bacteria. A bacteria as described herein, wherein said bacteria is a Clostridium, Clostridium acetobutylicum, Clostridium acetobutylicum M5, Clostridium thermocellum, Clostridium ljungdahlii, Clostridium thermoautotrophicum, or Clostridium tyrobutyricum. A bacteria as described herein, wherein said bacteria is a Butyrobacterium, Moorella thermoacetica, Sporomusa, Thermacetogenium phaeum, Acetogenium kivui, Acetobacterium woodii, or Eubacterium. A bacteria as described herein, The bacteria of claim 1, wherein said bacteria is Clostridium acetobutylicum M5. A method for the bioproduction of a C4+ compound, said method comprising: anaerobically growing a bacteria as described herein in a culture medium comprising a carbon source; conversion of said carbon source to a C4+ compound; and purification of said C4+ compound. A method as herein described, wherein said C4+ compound is butyrate, butanol, valeric acid, pentanol, hexanoate, hexanol, heptanoate, heptanol, octanoate, octanol, or a derivative thereof, and is preferably, butyrate, butanol or derivatives thereof A method as herein described, wherein said growing step is anaerobic. A method as herein described wherein said growing step is 100% anaerobic. A method as herein described, wherein said growing step is <100% anaerobic, e.g., the bacteria is not a strict anaerobe. Such bacteria can be selected for some degree of oxygen tolerance before or after the genetic engineering step.

Materials and Methods

(16) Experiments were performed in anaerobic glove box containing 85% N.sub.2, 10% H.sub.2 and 5% CO.sub.2 atmosphere. Glycerol stocks of C. acetobutylicum ATCC 824 and its mutant strain M5 (Clark 1989) harboring pSOS94-FNR (FIG. 3) or pJIR750-FNR (FIG. 4) were streaked on 2YTG and incubated at 37 C. for 2-5 days. Preculture was prepared by inoculating a single colony from 2YTG plate in 10 mL of CGM+ containing 25 mg/L thiamphenicol or 40 mg/L erythromycin in 15 mL falcon tube and incubated at 37 C. for 14-16 h. 200 L of preculture was used as inoculum for fermentation experiments performed in 15 mL tubes with loose cap containing 10 mL CGM+ with 50 g/L glucose at 37 C. without shaking.

(17) Samples were collected at various time points to measure OD600 and metabolites. 1 mL sample was centrifuged at 12,000 rpm for 5 min at room temperature to remove cell debris and clear supernatant was acidified with 20 L 50% H.sub.2SO.sub.4. Metabolites such as ethanol, acetone, acetic acid, butanol and butyric acid were measured by gas chromatography equipped with FID detector and PoraPak QS 80/100 glass column.

(18) In the Tables below, ATCC824 is wild type Clostridium acetobutylicum. M5 is a pSOL1.sup. mutant strain of the same bacterium. This mutant is used to show that the redistribution of redox from reduced Fd can generate longer chain acids in the metabolite profile of an acidogenic culture, and would be similar to the acidigenic metabolites produced by Clostridium tyrobutyricum or clostridium butyricum, as an example of the effect on a non-solvent producing clostridium species. The A and B refer to different isolates from the same transformation.

(19) pJIR750 is a Clostridium perfringens-Escherichia coli shuttle vector derived from pJIR418 (ATCC 77387) permitting expression of antibiotic resistance (chloramphenicol resistance (catP)) in both hosts. pJIR750-FNR contains a codon optimized gene for FNR (SEQ ID NO. 2).

(20) TABLE-US-00005 TABLE 1 butyrate/acetate ratio of wild type C. acetobutylicum with or without FNR from Chlorobium tepidium Butyrate:Acetate ratio Strain 6 h 24 h 48 h 72 h ATCC824(pJIR750)_A 0.72 0.40 0.51 0.47 ATCC824(pJIR750)_B 1.11 0.50 0.46 0.37 ATCC824(pJIR750-FNR)_A 1.53 0.65 0.86 1.13 ATCC824(pJIR750-FNR)_B 1.65 0.67 1.04 1.20

(21) TABLE-US-00006 TABLE 2 butanol/acetone ratio of wildtype C. acetobutylicum with or without FNR from Chlorobium tepidium Butanol:Acetone ratio Strain 6 h 24 h 48 h 72 h ATCC824(pJIR750)_A 14.78 1.55 1.50 1.55 ATCC824(pJIR750)_B 18.16 1.37 1.46 1.57 ATCC824(pJIR750-FNR)_A 29.52 2.23 2.36 2.70 ATCC824(pJIR750-FNR)_B 23.41 2.28 2.80 3.18

(22) TABLE-US-00007 TABLE 3 butyrate/acetate ratio of pSOL1.sup. mutant C. acetobutylicum with or without FNR from Chlorobium tepidium Butyrate:Acetate ratio Strain 6 h 24 h 48 h 72 h M5_A 1.21 2.15 2.21 2.03 M5_B 0.93 1.61 1.85 1.71 M5(pJIR750)_A 1.17 1.78 1.77 1.71 M5(pJIR750)_B 1.23 1.83 1.88 1.73 M5(pJIR750-FNR)_A 4.61 3.97 4.35 3.81 M5(pJIR750-FNR)_B 2.34 3.45 3.65 3.15

(23) The following experiment was done in a mutant of Clostridium acetobutylicumPJC4BKthat is disrupted in buk, the gene encoding the major butyrate kinase, and called BUK or buk.sup. herein. This strain has higher butanol formation than the parent ATCC824 because it is limited for the pathway from butyryl-CoA to butyrate as described in Green (1996). It was used as a host for the same plasmids described above in order to show the effect in a higher solvent forming derivative of C. acetobutylicum that already produces a higher level of butanol than the wild type parent.

(24) pJIR750-FNR has a SalI fragment of pSOS94-FNR subcloned into pJIR750. pSOS94-FNR contains codon optimized Ferredoxin NAD(P) reductase of Chlorobium tepidium (DNA2.0 construct). pSOS94 is another shuttle vector for Clostridia, but the FNR was moved because the pSOS vector and the chromosome of the high solvent producing mutant both carry the MLS drug resistance marker for erythromycin. Thus, the use of a shuttle vector with a different selectable marker was needed.

(25) A single colony from transformation plate (2YTG agar plate with Thiamphenicol 20 g/ml) inoculated in 10 mL CGM+ medium containing Th2018 h at 37 C. (CGM+ is a modified CGM recipe with 50 g/L glucose) 100 l of preculture inoculated in 10 mL CGM+ in 15 ml tube+Th2037 C. for 72 hours. 1 ml sample collected at 6 h, 24 h, 48 h and 72 hcentrifuged, acidified with 20 L 50% H.sub.2SO.sub.4 and ran on GC for metabolites and the results shown in Tables 4-7.

(26) The experimental is as described in the previous examples.

(27) Table 4 shows the addition of the plasmid with the FNR.sup.+ does not drastically effect the growth or OD value. The control Buk.sup.JIR750 is with a plasmid alone with no FNR gene in it. The test strain has FNR encoded by the same vector. A and B are two different isolates from the same transformation.

(28) TABLE-US-00008 TABLE 4 OD of the cultures Buk.sup. JIR750 and Buk.sup. JIR750-FNR.sup.+ Strain Short name 6 hr 24 hr 48 hr Buk.sup. JIR750 - A Control-1 0.7 12.54 11 Buk.sup. JIR750 - B Control-2 0.6 12.16 12.4 Buk.sup. JIR750-FNR.sup.+ - A FNR-1 0.5 10.64 11.6 Buk.sup. JIR750-FNR.sup.+ - B FNR-2 0.5 9.12 11

(29) The metabolite analysis of the fermentation products of the cultures is seen in Table 5, where butanol production is considerably improved.

(30) TABLE-US-00009 TABLE 5 Metabolite levels in BUK Clostridium with or without FNR Buk host plus vector alone = control-1 and control 2 Buk(pJIR750-FNR) = FNR-1 & FNR-2 mM of metabolite Control-1_48 h Ethanol 39.938 Acetone 47.160 Acetate 10.382 Butanol 95.580 Butyrate 12.730 Control-2_48 h Ethanol 39.706 Acetone 49.680 Acetate 9.730 Butanol 97.840 Butyrate 14.350 FNR-1_48 h Ethanol 36.705 Acetone 30.460 Acetate 6.080 Butanol 102.290 Butyrate 6.220 FNR-2_48 h ethanol 33.966 acetone 33.550 acetate 5.640 butanol 104.420 butyrate 6.590

(31) Looking at important ratios for high butanol production processes, the effect of the added enzyme is significant and positive, as seen in Tables 6-8.

(32) TABLE-US-00010 TABLE 6 Solvent/acid ratio metabolite levels in BUK Clostridium with or without FNR. The solvents are a total of butanol plus acetone plus ethanol the acids are acetate and butyrate 24 h 48 h Control-1 8.533 7.904 Control-2 8.329 7.775 FNR-1 12.150 13.777 FNR-2 11.362 14.059

(33) TABLE-US-00011 TABLE 7 % Butanol of total solvents (g/L basis) in BUK Clostridium with or without FNR Strains 24 h 48 h 72 h Control-1 56.708 60.741 65.013 Control-2 56.82 60.6 64.136 FNR-1 63.687 68.664 72.274 FNR-2 63.494 68.778 73.057

(34) TABLE-US-00012 TABLE 8 Acetone and Butanol with FNR from C. tepidium in a BUK background Butanol Butanol/ Butanol/ Acetone (mM) (mM) acetone acetone Strain 72 hrs 72 hrs 48 hrs 72 hrs buk pJIR750 41.8 102 2.0 2.44 buk pJIR750-FNR 24.7 106 3.23 4.32

(35) The butanol mass proportion is shown in Table 9, and butanol/solvent in Table 10.

(36) TABLE-US-00013 TABLE 9 Butanol mass proportion with FNR from C. tepidium in a BUK background Butanol as % of total Butanol as Butanol as metabolites % of total % of total (g/L) metabolites (g/L) metabolites (g/L) Strain 24 hrs 48 hrs 72 hrs buk pJIR750 51.6 54.3 58.3 buk pJIR750-FNR 60.8 65.8 69.9

(37) TABLE-US-00014 TABLE 10 Butanol mass solvent proportion with FNR from C. tepidium in a BUK background Butanol as Butanol as Butanol as % of total % of total % of total solvents (g/L) solvents (g/L) solvents (g/L) Strain 24 hrs 48 hrs 72 hrs buk pJIR750 59.3 62.6 66.9 buk pJIR750-FNR 66.8 71.4 75.5

(38) These experiments show that the proportion of butanol and butyrate is increased in the presence of the FNR gene encoded by C. tepidium.

(39) Although one might predict that any enzyme that reduces NAD to NADH could be used herein, our experimental work shows this is not in fact so. TER uses NADH directly to reduce the crotonyl-CoA to butyryl-CoA and has been used in E. coli to make butanol. However, our results show that it will not function as intended herein.

(40) A different TER was tested and also found to be non-functional, suggesting that the above result is generally applicable. The gene coding for the T. denticola enzyme (Acc. No. NP_971211.1) was synthesized, expression optimized by DNA2.0, cloned into the vector pJIR750 and transformed into C. acetobutylicum M5 (a non-solvent producing strain) so acetate vs butyrate can be examined. Table 11 shows a reduction of butyrate, rather than an improvement.

(41) TABLE-US-00015 TABLE 11 Acetate and Butyrate with TER from Treponema denticola in pSOL.sup. background Acetate MM Butyrate (mM) butyrate/acetate ratio STRAIN 24 HR 24 hr 24 hr M5 pJIR750 24.9 44.7 1.8 OD = 5.5 M5 pJIR750-TER 22.4 33.5 1.5 OD = 5.9 Acetone (mM) Butanol (mM) butanol/acetone ratio 72 hrs 72 hrs 72 hr M5 pJIR750 47.8 74.7 1.56 M5 pJIR750-TER 25.8 49.4 1.91

(42) The TER experiments were another way of doing the NADH utilization as this enzyme reduced the crotonyl-CoA to butyryl-CoA with just NADH rather than the Clostridial enzyme that uses NADH and oxidized Fd and gives butyryl-CoA and reduced Fd and NAD in a more complex bifurcating reaction. It was synthesized and expressed in the same way as the FNR enzyme, but it had a lowering effect on the butyrate and butanol in the way it was expressed and tested. These negative results suggest that the minimum requirement for functionality hereunder is addition of extra ferredoxin NAD(P)H reductase activity. We did not test TER in the presence of FNR to see if the coupled system had a positive effect.

(43) Each of the following citations is incorporated by reference herein in its entirety for all purposes: Cai, X., et al., 2013. Analysis of redox responses during TNT transformation by Clostridium acetobutylicum ATCC 824 and mutants exhibiting altered metabolism. Appl Microbiol Biotechnol. 97(10):4651-4663. Calusinska, M., et al., 2010. The surprising diversity of clostridial hydrogenases: a comparative genomic perspective. Microbiology. 156(6): 1575-1588. Clark, S. W., at al., 1989. Isolation and characterization of mutants of Clostridium acetobutylicum ATCC 824 deficient in acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase (EC 2.8.3.9) and in other solvent pathway enzymes. Appl. Environ Microbiol. 55 (4): 970-976. Frigaard, N. U., et al., 2003. Chlorobium tepidum: insights into the structure, physiology, and metabolism of a green sulfur bacterium derived from the complete genome sequence. Photosynth Res. 78(2):93-117. Girbal, L., et al., 2005. Homologous and heterologous overexpression in Clostridium acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases with high specific activities. Appl. Environ Microbiol. 71(5):2777-2781. Green, E. M., et al., 1996. Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824, Microbiology, 142, 2079-2086. Hillmann F, et al., 2008. PerR acts as a switch for oxygen tolerance in the strict anaerobe Clostridium acetobutylicum, Mol Microbiol. 68(4):848-60. Hurley J. K., et al., 2002. Structure-function relationships in Anabaena ferredoxin/ferredoxin:NADP(+) reductase electron transfer: insights from site-directed mutagenesis, transient absorption spectroscopy and X-ray crystallography, Biochim Biophys Acta. 1554(1-2):5-21. Peters, J. W., et al., 1998. X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution. Science. 282(5395):1853-1858. Peregrina J. R., et al., 2009. Motifs involved in coenzyme interaction and enzymatic efficiency in anabaena ferredoxin-NADP+ reductase. Biochemistry. 2009 48(14):3109-19. Seo, D., Sakurai, H. 2002. Purification and characterization of ferredoxin-NAD(P)(+) reductase from the green sulfur bacterium Chlorobium tepidum. Biochim Biophys Acta. 1597 (1):123-132. Seo, D., et al., 2001. Purification of ferredoxins and their reaction with purified reaction center complex from the green sulfur bacterium Chlorobium tepidum. Biochim Biophys Acta. 1503(3):377-384. Tejero J. et al., 2003. Involvement of the pyrophosphate and the 2-phosphate binding regions of ferredoxin-NADP+ reductase in coenzyme specificity, J Biol Chem. 278(49):49203-14. Wagner C., et al., 1996. Acetogenic capacities and the anaerobic turnover of carbon in a kansas prairie soil, Appl. Environ. Microbiol. 62(2): 494-500. Watrous, M. M., et al., 2003. 2,4,6-trinitrotoluene reduction by a Fe-only hydrogenase in Clostridium acetobutylicum. Appl. Environ Microbiol. 69(3):1542-1547. Yoon, K. S., et al., 2001. Spectroscopic and functional properties of novel 2[4Fe-4S] cluster-containing ferredoxins from the green sulfur bacterium Chlorobium tepidum. J. Biol. Chem. 276(47):44027-44036. Shen et al. 2011. Driving forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli. Appl Environ Microbiol. 77(9):2905-15.