Reverse beta oxidation pathway

Abstract

The invention relates to recombinant microorganisms that have been engineered to produce various chemicals using genes that have been repurposed to create a reverse beta oxidation pathway. Generally speaking, the beta oxidation cycle is expressed and driven in reverse by modifying various regulation points for as many cycles as needed, and then the CoA thioester intermediates are converted to useful products by the action of termination enzymes.

Claims

1. A method of making a product, comprising growing an engineered microorganism in a nutrient broth for a time sufficient to make a product, and isolating said product, wherein said product is selected from the group consisting of carboxylic acids, alkanes, or alkenes, wherein said engineered microorganism comprises: a) overexpression of -oxidation cycle enzymes as compared to a corresponding wild type microorganism, wherein said enzymes comprise: i) a thiolase catalyzing the conversion of (C.sub.n)-acyl CoA to -ketoacyl-CoA; ii) a hydroxyacyl-coA dehydrogenase catalyzing the conversion of -ketoacyl-CoA to -hydroxyacyl-CoA; iii) an enoyl-coA hydratase catalyzing the conversion of -hydroxyacyl-CoA to trans-2-enoyl-coA; and iv) an acyl-CoA dehydrogenase or a transenoyl-CoA reductase catalyzing the conversion of trans-2-enoyl-coA to (C.sub.n+2)-acyl CoA; b) functional operation of a -oxidation cycle in a reverse biosynthetic direction as recited in steps i) to iv); and c) overexpression of one or more termination enzyme(s) as compared to a corresponding wild type microorganism, wherein said termination enzyme(s) are selected from: i) an alcohol-forming coenzyme-A thioester reductase, or an aldehyde-forming CoA thioester reductase plus an alcohol dehydrogenase, to convert intermediates produced by reversal of the -oxidation cycle to trans 2 fatty alcohols, -keto alcohols, 1,3 diols, or -hydroxy acids; ii) a thioesterase, or an acyl-CoA:acetyl-CoA transferase, or a phosphotransacylase and a carboxylate kinase, to convert intermediates produced by reversal of the -oxidation cycle to carboxylic acids; iii) an aldehyde-forming CoA thioester reductase and an aldehyde decarbonylase, to convert intermediates produced by reversal of the -oxidation cycle to alkanes or terminal alkenes; and iv) one or more olefin-forming enzymes to convert intermediates produced by reversal of the -oxidation cycle to alkenes.

2. The method of claim 1, further comprising supplementing said nutrient broth with propionate in order to produce odd-chain length products.

3. The method of claim 1, further comprising growing said engineered microorganism under microaerobic (<10% O.sub.2) or anaerobic conditions at a temperature of 30-40 C., wherein said nutrient broth comprises 0-100 M FeSO.sub.4 and 0-5 mM calcium pantothenate.

4. The method of claim 1, wherein said one or more olefin-forming enzymes are selected from: OleA, OleB, OleC, and OleD.

5. The method of claim 4, wherein said engineered microorganism comprises a genotype selected from the group consisting of: a) fadR, atoC(c), arcA, crp, crp*, adhE, frdA, pta, yqhA, fucO, fadD, ydiO, [yqeF+, tesB+]; b) fadR, atoC(c), arcA, crp, crp*, adhE, frdA, pta, yqhA, fucO, fadD, [fadBA+, fadM+]; and c) fadR, atoC(c), arcA, crp, crp*, adhE, frdA, pta, yqhA, fucO, fadD, [fadBA+, yciA+].

6. The method of claim 4, wherein said engineered microorganism comprises a genotype selected from the group consisting of: a) fadR, atoC(c), arcA, crp, crp*, adhE, frdA, pta, yqhA, fucO, fadD, [yqeF+, acrM+, PCC7942_orf1593+]; b) fadR, atoC(c), arcA, crp, crp*, adhE, frdA, pta, yqhA, fucO, fadD, [yqeF+, acrM+, PCC7942_orf1593+]; c) fadR, atoC(c), arcA, crp, crp*, adhE, frdA, pta, yqhA, fucO, fadD, [yqeF+, oleABCD+,]; d) fadR, atoC(c), arcA, crp, crp*, adhE, frdA, pta, yqhA, fucO, fadD, [fadBA+, acrM+, PCC7942_orf1593+]; e) fadR, atoC(c), arcA, crp, crp*, adhE, frdA, pta, yqhA, fucO, fadD, [fadBA+, acr1+, PCC7942_orf1593+]; and f) fadR, atoC(c), arcA, crp, crp*, adhE, frdA, pta, yqhA, fucO, fadD, [fadBA+, oleABCD+,].

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A Proposed metabolic platform for the combinatorial synthesis of advanced fuels and chemicals using a functional reversal of the -oxidation cycle. The engineered reversal of the -oxidation cycle reported in this work is composed of the following enzymes (gene names in parentheses): {circle around (1)} thiolase (yqeF, fadA, atoB); {circle around (2)} hydroxyacyl-CoA dehydrogenase (fadB, fadJ); {circle around (3)} enoyl-CoA hydratase (fadB, fadJ) (note: {circle around (2)} and {circle around (3)} are fused in this species); {circle around (4)} acyl-CoA dehydrogenase (ydiO, fadE). Each turn of the reverse cycle generates an acyl-CoA that is two carbons longer than the initial acyl-CoA thioester (indicated as C.sub.n+2). Intermediates in the engineered pathway can be converted to a functionally diverse set of alcohols and carboxylic acids of different chain lengths using one or more of i) alcohol-forming CoA thioester reductases or ii) aldehyde-forming CoA thioester reductases and alcohol dehydrogenases ({circle around (5)}) or iii) CoA thioester hydrolases/thioesterases, or acyl-CoA:acetyl-CoA transferases, or phosphotransacylase and carboxylate kinases ({circle around (6)}), as indicated. Products whose synthesis was demonstrated in this study are shown in boxes. Abbreviations: R indicates the side chain (e.g. RH for acetyl-CoA and RCH.sub.3 for propionyl-CoA) attached to the acyl-CoA group of the primer or starter molecule. Dotted lines indicate multiple steps while dashed lines without arrowheads connect identical metabolites of different chain length. A comparison of n-alcohol production between the reversal of the -oxidation cycle engineered in this work and the recently proposed fatty acid biosynthesis pathway.sup.2 is shown in FIG. 4.

(2) FIG. 1B Regulation of operons encoding the proposed reversal of the -oxidation cycle by regulators FadR, ArcAB, FNR, CRP-cAMP, AtoSC. Activation and repression of operons is indicated by and , respectively.

(3) FIG. 2a-c Engineered one-turn reversal of the -oxidation cycle for the synthesis of n-butanol and short-chain carboxylic acids. FIG. 2(a) Effect of gene overexpressions and knockouts (indicated underneath x axis) on the synthesis of n-butanol and ethanol in strain RB02 (fadR atoC(c) crp* arcA adhE pta frdA). Experiments were performed at 30 C. for 24 hours in shake flasks using glucose (1% w/v) minimal medium. The n-butanol yield was calculated as g n-butanol/g total glucose consumed. FIG. 2(b) Kinetics of n-butanol production by strain RB02 yqhD eutE [yqeF+fucO+]. Cells were cultivated in fermentors containing minimal medium supplemented with 5% (w/v) glucose. The dissolved oxygen was controlled at 5% of saturation, temperature at 30 C., and pH at 7. FIG. 2(c) Synthesis of -ketobutyric (left panel), -hydroxybutyric (center panel), and trans-2-butenoic (right panel) acids upon overexpression of thioesterases I (TesA) and II (TesB) in strains RB02, RB02fadB, and RB02ydiO. Experiments were run at 37 C. for 48 hours in shake flasks using glucose (1% w/v) minimal medium.

(4) FIG. 3(a)-(d): Synthesis of higher-chain (C>4) carboxylic acids (FIGS. 3a and b) and n-alcohols (FIGS. 3c and d) through the engineered reversal of the b-oxidation cycle. FIG. 3(a) Accumulation of long-chain (C>10) free fatty acids in the extracellular medium of strain RB03 [fadBA+] upon overexpression of different thioesterases (FadM, YciA, TesA, TesB). Product yield is shown above the bars (g free fatty acid/g total glucose consumed100). Experiments were run at 37 C. for 48 hours in shake flasks using glucose (2% w/v) minimal medium. FIG. 3(b) Kinetics of fatty acid synthesis by strain RB03 [fadBA,fadM+]. Cells were cultivated in fermentors using glucose (3% w/v) minimal medium. The dissolved oxygen was controlled at 2% of saturation, temperature at 37 C., and pH at 7. FIG. 3(c) Synthesis of n-alcohols in strain RB03 [fadBA+] upon overexpression of alcohol dehydrogenases (YiaY, BetA, and EutG). Product yield is shown above the bars (g n-alcohol/g total glucose consumed100). Experiments were run at 37 C. for 48 hours in shake flasks using glucose (2% w/v) minimal medium. FIG. 3(d) Effect of alcohol dehydrogenase overexpression (YiaY, BetA, and EutG) on the chain-length distribution of n-alcohols synthesized by strain RB03 [fadBA+] in the presence of 0.5 g/L propionate. Experiments were conducted as described in panel c.

(5) FIG. 4 is a comparison of n-alcohol synthesis via the fatty acid biosynthesis pathway (top) and the engineered reversal of the -oxidation cycle (bottom). Reactions {circle around (1)}-{circle around (5)} are as indicated in FIG. 1. The fatty acid biosynthesis pathway uses acyl-ACP intermediates and involves a -ketoacyl-acyl-carrier protein synthase ({circle around (8)}) along with a -ketoacyl reductase, an enoyl reductase, and a -hydroxyacyl dehydratase ({circle around (9)}). The synthesis of malonyl-ACP, the 2-C donor during chain elongation in fatty acid biosynthesis, is also shown ({circle around (7)}). Production of n alcohols from these acyl-ACP intermediates requires their conversion to free acids ({circle around (10)}) and acylation ({circle around (11)}) before their reduction to alcohols ({circle around (6)}) can be achieved. The use of acyl-ACP intermediates and malonyl-ACP as the 2-C donor during chain elongation in the fatty acid biosynthesis pathway limits its ATP efficiency, making it an ATP-consuming pathway, as shown in the following balanced equation for n-alcohol synthesis from glucose:
n/4C.sub.6H.sub.12O.sub.6+ATP.fwdarw.C.sub.nH.sub.n+2O+n/2CO.sub.2+(n/21)H.sub.2O,

(6) with n being the chain length of the n-alcohol.

(7) FIG. 5 is synthesis of hydrocarbons through engineered reversal of the -oxidation cycle and efficient operation of the core pathway by optimal coupling of generation and consumption of reducing equivalents. Reaction are as indicated in FIG. 1. Reaction indicates the synthesis of alkanes from acyl-CoAs as shown in FIG. 1A. Reaction indicates the synthesis of alkenes from acyl-CoAs via the head-to-head condensation mechanism shown in FIG. 1B. 2[H]=NADP(H)=FAD(H).sub.2=Fd.sub.red. Termination pathways 5 and 6 enable the synthesis of hydrocarbons from CoA-thioester intermediates using aldehyde-forming CoA thioester reductases and aldehyde decarbonylases (pathway 5, leading to the formation of alkanes or terminal alkenes of different chain lengths) and olefin-forming enzymes (pathway 6, leading to the formation of aliphatic internal alkenes or terminal alkenes or trienes or alkenols). Also noted are dissimilation of pyruvate through routes that preserve reducing equivalents (as opposed to releasing them in the form of hydrogen: e.g. PDH*, PNO, YdbK, NADH-dependent FDH), use of NAD(P)H-dependent trans-enoyl-CoA reductases (reaction 4), and direct coupling between trans-enoyl-CoA reduction (reaction 4) and pyruvate oxidation.

(8) FIG. 6 is a diagram illustrating the map of plasmid pZS acrM synpcc7942_1593. This plasmid expresses codon optimized Acinetobacter acrM gene (encoding an aldehyde-forming CoA thioester reductase) and codon optimized Synechococcus synpcc7942_1593 gene (encoding an aldehyde decarbonylases). These two enzymes form a termination pathway that leads to the formation of alkanes or terminal alkenes of different chain lengths from the CoA-thioester intermediates of the -oxidation reversal.

(9) FIG. 7A-B contains diagrams illustrating the maps of plasmids pZS Egter (A) and pZS Tdter (B), carrying the genes that encode E. gracilis and T. denticola NAD(P)H-dependent transenoyl-CoA dehydrogenases, respectively.

(10) FIG. 8A-C contains diagrams illustrating the maps of plasmids pZS ydbK (A) pZS ydbKydiQRST (B), and pZS ydiO (C). These plasmids carry the genes that code for E. coli pyruvate:flavodoxin oxidoreductase (YdbK), acyl-CoA dehydrogenase (YdiO) and required electron transfer flavoproteins and ferredoxin (YdiQRST).

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

(11) We have engineered a functional reversal of the fatty acid oxidation cycle (aka -oxidation) in E. coli and used it in combination with endogenous dehydrogenases and thioesterases to produce n-alcohols and fatty acids of different chain lengths (FIG. 1).

(12) The engineered pathway operates with coenzyme-A (CoA) thioester intermediates and uses acetyl-CoA directly for acyl-chain elongation (rather than first requiring ATP-dependent activation to malonyl-CoA), features that enable product synthesis at maximum carbon and energy efficiency.

(13) The synthesis of substituted and unsubstituted n-alcohols and carboxylic acids (FIGS. 2 and 3) at yields and titers higher than previously reported demonstrate the superior nature of the engineered pathway. The ubiquitous nature of the -oxidation cycle should enable the efficient synthesis of a host of non-native products in industrial organisms without recruiting foreign genes, an approach we term here homologous metabolic engineering.

(14) We will shortly demonstrate the production of alkanes and alkenes using non-native enzymes added to the bacteria, and the vectors for same have already been constructed.

Example 1: Materials and Methods

(15) The material and methods detailed herein are exemplary only, but the techniques are standard in the art and different methodologies can be substituted herein. What is important is the engineering to effect pathway reversal, direct carbon flow, and upregulating the termination enyzmes.

(16) Reagents

(17) Chemicals were obtained from FISHER SCIENTIFIC (Pittsburgh, Pa.) and SIGMA-ALDRICH CO. (St. Louis, Mo.).

(18) Culture Medium

(19) The minimal medium designed by Neidhardt (1974) with Na.sub.2HPO.sub.4 in place of K.sub.2HPO.sub.4 and supplemented with 20 g/L glucose, 40 g/L calcium bicarbonate, 100 M FeSO.sub.4, 5 mM calcium pantothenate, 3.96 mM Na.sub.2HPO.sub.4, 5 mM (NH.sub.4).sub.2SO.sub.4, and 30 mM NH.sub.4Cl was used. Fermentations conducted in the SIXFORS multi-fermentation system also included 1 mM betaine.

(20) Plasmid Construction

(21) Standard recombinant DNA procedures were used for gene cloning, plasmid isolation, and electroporation. Manufacturer protocols and standard methods were followed for DNA purification (QIAGEN, CA, USA), restriction endonuclease digestion (NEW ENGLAND BIOLABS, MA, USA), and DNA amplification (STRATAGENE, CA, USA and INVITROGEN, CA, USA). For plasmid construction, genes were amplified from MG1655 genomic DNA using primers designed to create 15 bp of homology on each end of the gene insert for subsequent recombination into the desired plasmid. Plasmids were linearized using restriction endonuclease digestion, then recombined with the appropriate gene(s) using an IN-FUSION DRY-DOWN PCR CLONING KIT (CLONTECH, Mountain View, Calif., USA) and subsequently used to transform chemically competent FUSION BLUE cells (CLONTECH, Mountain View, Calif., USA).

(22) Transformants that grew on LB plates containing the appropriate antibiotic were struck for isolation, and then subjected to preliminary screening by PCR. Colonies passing preliminary inspection were then individually grown for plasmid purification. Purified plasmids were confirmed to have the appropriate insert both by PCR as well as restriction endonuclease digest verification. Plasmids in each case include the plasmid promoter, a ribosomal binding site for each gene, MG1655 gene(s), and a plasmid terminator. Resulting plasmids (and strains) are listed in Tables 3 and 4.

(23) Metabolite Identification

(24) The identity of metabolic products was determined through one-dimensional (1D) proton nuclear magnetic resonance (NMR) spectroscopy. 60 L of D.sub.2O and 1 L of 600 mM NMR internal standard TSP [-(trimethylsilyl) propionic acid-D4, sodium salt] were added to 540 L of the sample (culture supernatant). The resulting solution was then transferred to a 5 mm-NMR tube, and 1D proton NMR spectroscopy was performed at 25 C. in a Varian 500-MHz Inova spectrometer equipped with a Penta probe (VARIAN, INC., Palo Alto, Calif.) using the following parameters: 8,000-Hz sweep width, 2.8-s acquisition time, 256 acquisitions, 6.3-s pulse width, 1.2-s pulse repetition delay, and presaturation for 2 s. The resulting spectrum was analyzed using FELIX 2001 software (ACCELRYS SOFTWARE INC., Burlington, Mass.). Peaks were identified by their chemical shifts and J-coupling values, which were obtained in separate experiments in which samples were spiked with metabolite standards (2 mM final concentration).

(25) Identification of n-alcohols was conducted through gas chromatography-mass spectroscopy (GC-MS) following a modification of the method reported by Atsumi (2008). The analysis was performed on an AGILENT 6890 GC/5973 MS (AGILENT TECHNOLOGIES, Palo Alto, Calif.) instrument with a HP-5 ms capillary column (30 m0.25 mm0.25 m). 1 ml of supernatant of culture broth was extracted with 500 l of GC standard grade hexane (Fluka). 0.5 l of the extracted sample was injected using a 20:1 split at 250 C. The oven temperature was initially held at 75 C. for 2 min and then raised with a gradient of 5 C./min to 280 C. and held for 2 min. Helium (MATHESON TRI-GAS, Longmont, Colo.) was used as the carrier gas with a 14-1b/in.sup.2 inlet pressure. The injector and detector were maintained at 255 C.

(26) Identification of fatty acids was performed on a SHIMADZU Auto-System GC 2010 (SHIMADZU, Japan) equipped with a DB-5MS capillary column (30 m0.25 mm0.25 m) and directly connected to MS. The following method was used: an initial temperature of 50 C. was held for 2 min and then ramped to 220 C. at 4 C. per min and held for 10 min.sup.2. Extraction and derivatization procedures are described in section Metabolite Quantification.

(27) Metabolite Quantification

(28) The quantification of glucose, organic acids, ethanol, and butanol was conducted by high-performance liquid chromatography (HPLC). Samples (culture supernatant) were analyzed with ion-exclusion HPLC using a SHIMADZU Prominence SIL 20 system (SHIMADZU SCIENTIFIC INSTRUMENTS, INC., Columbia, Md.) equipped with an HPX-87H organic acid column (BIO-RAD, Hercules, Calif.) with operating conditions to optimize peak separation (0.3 mL/min flowrate, 30 mM H.sub.2SO.sub.4 mobile phase, column temperature 42 C.).

(29) Quantification of longer chain (C4) n-alcohols was conducted through gas chromatography (GC) in a VARIAN CP-3800 gas chromatograph (VARIAN ASSOCIATES, INC., Palo Alto, Calif.) equipped with a flame ionization detector (GC-FID). Sample extraction procedure was as described above in section Metabolite Identification. The separation of alcohol compounds was carried out using a VF-5ht column (15 m, 0.32 mm internal diameter, 0.10 m film thickness; VARIAN ASSOCIATES, INC., Palo Alto, Calif.). The oven temperature was initially held at 40 C. for 1 min and then raised with a gradient of 30 C./min to 130 C. and held for 4 min. The temperature was then raised with a gradient of 15 C./min to 230 C. and held for 4 min. Helium (1 ml min.sup.1, MATHESON TRI-GAS, Longmont, Colo.) was used as the carrier gas. The injector and detector were maintained at 250 C. A 0.5-l sample was injected in splitless injection mode.

(30) Quantification of fatty acids was carried out in a VARIAN CP-3800 gas chromatograph (VARIAN ASSOCIATES, INC., Palo Alto, Calif.) after hexane-methyl tertiary butyl ether (MTBE) extraction (Lalman 2004) and FA transesterification with a mixture of cholophorm:methanol:hydrochloric acid [10:1:1, vol/vol/vol] as previously reported (Dellomonaco 2010). The resulting fatty acids methyl esters were quantified according to the following method: 50 C. held for 1 min, 30 C./min to 160 C., 15 C./min to 200 C., 200 C. held for 1.5 min, 10 C./min to 225 C., and 225 C. held for 15 min.

(31) Enzyme Assays

(32) For measurement of enzymatic activities, cells from 24 hour shake flask cultures were washed twice with 9 g/L sodium chloride under anaerobic conditions and stored at 80 C. until use. Cell extracts for all assays were prepared as follows under anaerobic conditions. 40 units of OD.sub.550 nm was re-suspended in 1 mL of 100 mM Tris-HCl buffer (pH 7.0) with 1 mM DTT. After cellular disruption using a DISRUPTOR GENIE (SCIENTIFIC INDUSTRIES, INC, Bohemia, N.Y.), cellular debris was removed by centrifugation (13,000g, 4 C., 10 min) and the supernatant used as cell extract. Absorbance changes for all assays were monitored in a BIOMATE 5 spectrophotometer (THERMO SCIENTIFIC, MA, USA). The linearity of reactions (protein concentration and time) was established for all assays and the non-enzymatic rates were subtracted from the observed initial reaction rates. Enzymatic activities are reported as mol of substrate per minute per mg of cell protein and represent averages for at least three cell preparations. Protein concentration was measured using the Bradford assay reagent (THERMO SCIENTIFIC, MA, USA) with BSA as a standard.

(33) Acetyl-CoA acetyltransferase (THL) activity was determined using acetoacetyl-CoA and CoA as substrates, and the decrease in acetoacetyl-CoA concentration was measured at 303 nm. -Hydroxybutyryl-CoA dehydrogenase activity was measured at 340 nm by monitoring the decrease in NADH concentration resulting from -hydroxybutyryl-CoA formation from acetoacetyl-CoA. Crotonase activity was measured by monitoring the decrease in crotonyl-CoA concentration at 263 nm, which results from -hydroxybutyryl-CoA formation from crotonyl-CoA. Butyryl-CoA dehydrogenase activity was assayed in the direction of crotonyl-CoA reduction by monitoring the ferricenium ion at 300 nm, which acts as an electron donor. In addition, assays in which the ferricenium ion was replaced with 0.2 mM NAD(P)H and the absorbance measured at 340 nm were also run. Butyraldehyde dehydrogenase activity was assayed in the direction of butyraldehyde oxidation by monitoring NAD(P).sup.+ reduction at 340 nm. To measure butanol dehydrogenase activity, the decrease in NAD(P)H concentration resulting from butanol formation from butyraldehyde is monitored at 340 nm under anaerobic conditions at 30 C.

Example 2: One-Turn Reversal of -Oxidation Cycle

(34) Given the applications of n-butanol as both advanced biofuels and building blocks for the chemical industry, we chose it as the first product to demonstrate the feasibility of engineering a functional reversal of the -oxidation cycle as an efficient platform for fuel and chemical production (FIG. 1).

(35) Synthesis of n-butanol can be realized through a one-turn reversal of the -oxidation cycle in combination with native aldehyde/alcohol dehydrogenases (FIG. 1a, reactions {circle around (1)}-{circle around (5)}). This engineered pathway represents an E. coli surrogate of the n-butanol pathway operating in Clostridia.

(36) Given the specificity of atoB-encoded acetyl-CoA acetyltransferase for short-chain acyl-CoA molecules and the high sequence similarity between atoB and yqeF (predicted acyltransferase), these genes were selected for Reaction {circle around (1)} of the pathway. The next two steps can be catalyzed by -hydroxyacyl-CoA dehydrogenases and enoyl-CoA hydratases, encoded by fadB and fadJ (Reactions {circle around (2)} and {circle around (3)} in FIG. 1a). The fourth step in this one-turn reversal of the -oxidation cycle can be catalyzed by acyl-CoA dehydrogenase (fadE or ydiO) (Reaction {circle around (4)}).

(37) The above genes are organized in four operons in the E. coli genome and are subjected to several levels of regulation (FIG. 1b). These regulatory pathways were therefore engineered to promote the reversal of the -oxidation cycle.

(38) Constitutive expression of fad and ato genes (regulated by FadR and AtoC, respectively: FIG. 1b) was achieved through fadR and atoC(c) mutations (Dellomonaco 2010). Since anaerobic/microaerobic conditions used in the production of fuels and chemicals would lead to repression of most target operons by ArcA (FIG. 1b), the arcA gene was also deleted.

(39) Several operons of interest are also activated by the cyclic-AMP receptor protein (CRP)-cAMP complex (FIG. 1b) and are therefore subjected to carbon catabolite repression in the presence of glucose. This regulatory mechanism was circumvented by replacing the native crp gene with cAMP-independent mutant crp* (Eppler & Boos, 1999). While these genetic manipulations were predicted to enable expression of the -oxidation cycle (FIG. 1b), no butanol synthesis was observed in strain fadR atoC(c) crp crp* arcA (RB01) or its parent fadR atoC(c) (Table 2). Table 5 provides details about mutations introduced at the crp, fadR, and atoSC loci.

(40) Given the significant accumulation of other fermentation products (Table 2), the pathways involved in the synthesis of ethanol, acetate, and succinate were also blocked (adhE, pta and frdA knockouts, respectively) in an attempt to channel carbon to the engineered pathway. Although the synthesis of these competing by-products was greatly reduced, strain RB02 (RB01 adhE pta frdA) did not produce n-butanol either (Table 2).

(41) Enzyme activity measurements confirmed a functional expression of the reversal of the -oxidation cycle in strain RB02, compared to negligible activity in wild-type MG1655 (Table 1a). However, the levels of n-butanol dehydrogenase were very low (Table 1a), probably preventing n-butanol synthesis.

(42) To address this issue, two endogenous aldehyde/alcohol dehydrogenases with high sequence and structure similarity to the Clostridial butyraldehyde/butanol dehydrogenase were overexpressed in strain RB02: i.e. L-1,2-propanediol oxidoreductase (fucO) and an aldehyde/alcohol dehydrogenase (yqhD) (Table 7). Despite the potential for YqhD to catalyze the conversion of butyraldehyde to n-butanol, overexpression of fucO led to higher n-butanol titer and yield (FIG. 2a). Nonetheless, both enzymes proved functional and either or both could be used.

(43) Although high levels of thiolase activity were observed in RB02 (Table 1a), these measurements account for enzymes with specificity for both short- and long-chain acyl-CoA molecules. In an attempt to divert acetyl-CoA to the n-butanol pathway, acetyl-CoA acetyltransferases that possess higher affinity for short-chain molecules (atoB and yqeF: FIG. 1a, Reaction {circle around (1)}) were overexpressed. The resulting strains, RB02 [atoB+] and RB02 [yqeF+], synthesized appreciable amounts of n-butanol (FIG. 2a).

(44) Overexpression of yqeF, whose function in E. coli metabolism is currently unknown, yielded higher concentrations of n-butanol and lower concentrations of the major fermentation by-product ethanol (FIG. 2a). No n-butanol synthesis was observed upon overexpression of atoB or yqeF in wild-type MG1655.

(45) Based on the above results, an increased partition of carbon flux towards n-butanol should be realized by simultaneous overexpression of yqeF, to channel acetyl-CoA into the engineered reversal of the -oxidation cycle, and fucO, to improve the conversion of butyryl-CoA to n-butanol. Indeed, strain RB02 [yqeF+fucO+] produced significant amounts of n-butanol (1.9 g/L) at a high n-butanol-to-ethanol ratio (>5:1) (FIG. 2a). No n-butanol production was observed upon simultaneous overexpression of yqeF and fucO in the wild-type background or in a strain containing pta, adhE, and frdA deletions, underscoring the importance of the fadR atoC(c) crp crp* arcA genotype.

(46) Since the engineering of strain RB02 [yqeF+fucO+] involved manipulation of several global regulators with potential pleiotropic effects, a characterization of the proposed reversal of the -oxidation cycle was conducted to establish its role on n-butanol synthesis (Table 1). Activity measurements showed high level of expression of key enzymes involved in the postulated pathway in strain RB02 [yqeF+fucO+] and negligible activity in wild type MG1655 (Table 1a). Gene knockout and gene complementation experiments along with quantification of fermentation products (Table 1b) demonstrated that the primary genes involved in the synthesis of n-butanol through the engineered one-turn reversal of the -oxidation pathway are (encoded activity in parenthesis): yqeF (predicted acyltransferase), fadB -hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase), ydiO (predicted acyl-CoA dehydrogenase), and fucO (L-1,2-propanediol oxidoreductase/n-butanol dehydrogenase).

(47) YdiO is proposed to catalyze the reduction of enoyl-CoA to acyl-CoA (Reaction {circle around (4)}). The reverse of this reaction is catalyzed by FadE and is the only irreversible step in the catabolic operation of the -oxidation cycle.sup.5. In agreement with our proposal, deletion of ydiO in strain RB02 [yqeF+fucO+] completely abolished n-butanol synthesis (Table 1b). Although ydiO was previously proposed to encode an acyl-CoA dehydrogenase that would replace FadE during the anaerobic catabolism of fatty acids.sup.20, a sequence comparison between YdiO and E. coli proteins does not reveal a significant similarity to FadE (Table 9). In contrast, YdiO shares high homology with crotonobetainyl-CoA reductase (CaiA). CaiA catalyzes the reduction of crotonobetainyl-CoA to -butyrobetainyl-CoA, a reaction similar to that catalyzed by YdiO in the reversal of the -oxidation cycle. Moreover, the operon fixABCX is required for the transfer of electrons to CaiA and encodes flavoproteins and ferredoxin with high sequence similarity to YdiQRST (Table 9). This analysis suggests that ferredoxin and flavoproteins encoded by ydiQRST are involved in the transfer of electrons to YdiO during the reduction of enoyl-CoA to acyl-CoA. Standard Gibbs energy calculations revealed that the engineered reversal of the -oxidation cycle is thermodynamically feasible if ferredoxin is the source of reducing power for the conversion of enoyl-CoA to acyl-CoA (Table 10). We then propose that the reduction of enoyl-CoA to acyl-CoA is mediated by YdiO-YdiQRST.

(48) Further reduction in the synthesis of by-product ethanol, and hence an increase in n-butanol yield, were realized by combining the overexpression of fucO and yqeF with the deletion of yqhD and eutE (aldehyde dehydrogenase with high sequence similarity to adhE). The resulting strain (RB02 yqhD eutE [yqeF+fucO+]) synthesized 2.2 g/L of n-butanol in 24 hours at a yield of 0.28 g n-butanol/g glucose (FIG. 2a). When grown in a bioreactor using a higher initial concentration of glucose, this strain produced n-butanol at high titer (14 g/L), yield (0.33 g n-butanol/g glucose) and rate (2 g n-butanol/g cell dry weight/h) (FIG. 2b).

(49) This performance, which was achieved without importing foreign genes and in the absence of rich nutrients, is an order of magnitude better than reported for any other organism engineered for n-butanol production and also surpasses the yield and specific productivity reported for native n-butanol producers. The reversal of the -oxidation cycle engineered in this strain operated at a maximum carbon flux of 73.4 mmol acetyl-CoA/g cell dry weight/h (12-18 hours in FIG. 2b), which exceeds the flux reported in the literature for native or engineered fermentative pathways. Taken together, these results demonstrate that the engineered reversal of the -oxidation pathway is a superior metabolic platform for the production of fuels and chemicals and can support the efficient synthesis of non-native products in industrial organisms without recruiting foreign genes (i.e. endogenous metabolic engineering).

(50) The engineered reversal of the -oxidation cycle generates a diverse set of CoA thioester intermediates that can be converted to the corresponding alcohols and carboxylic acids (FIG. 1A). To illustrate product synthesis from intermediates other than acyl-CoA, we used thioesterase I (TesA) and thioesterase II (TesB) as termination enzymes. Small amounts of -hydroxybutyric, -ketobutyric, and trans-2-butenoic acids were produced when these thioesterases were overexpressed in strain RB02 (FIG. 2c). The level of these products was significantly increased by simultaneous overexpression of thioesterase and yqeF-encoded short-chain acyltransferase (FIG. 2c). Further increases in product titer were realized upon deletion of fadB (500 mg/L -ketobutyric acid) and ydiO (150 mg/L and 200 mg/L of -hydroxybutyric and trans-2-butenoic acids, respectively) (FIG. 2c).

Example 3: Making Longer Chains (C>4)

(51) The operation of multiple cycles of the engineered reversal of the -oxidation pathway, and hence the synthesis of CoA-thioester intermediates (and products) of longer chain length (C4), can be facilitated by the overexpression of FadA, a -ketoacyl-CoA thiolase that is part of the -oxidation complex (FadBA) and which possesses broad chain length specificity.

(52) Overexpression of FadBA in conjuction with thioesterases (TesA, TesB, FadM or YciA) in strain RB03 (RB02yqhDfucO fadD) resulted in the accumulation of long-chain fatty acids in the extracellular medium (FIG. 3a). The fadD knockout in strain RB03 prevents re-utilization of the synthesized fatty acids. The choice of thioesterase allowed control over both length and functionality of the fatty acid side chain. For example, C16 and C18 saturated fatty acids were the only products when FadM was overexpressed while YciA and TesA overexpression supported the synthesis of -hydroxy (C14:3OH) and unsaturated (C18:1) fatty acids, respectively (FIG. 3a).

(53) When grown in a bioreactor using a higher initial concentration of glucose, strain RB03 [fadBfadM+] produced long-chain extracellular fatty acids at high titer (7 g/L) and yield (0.28 g fatty acids/g total glucose consumed) using a mineral salts medium without rich nutrients (FIG. 3b). These results are better than reported previously using an engineered fatty acid biosynthesis pathway (Table 9). No production of extracellular free fatty acids was observed upon overexpression of FadM in strain MG1655 adhE pta frdA fadD (Table 6C), demonstrating the requirement of an active reversal of the -oxidation cycle. Measurements of total free fatty acids (i.e. extracellular+intracellular) in strain RB03 [fadBfadM+] and the corresponding controls showed that the engineered reversal contributed to the synthesis of 90-95% of the total free fatty acids (Table 6C).

(54) The synthesis of longer-chain (C>4) n-alcohols was also demonstrated by overexpressing the appropriate termination enzymes (FIG. 3c). We identified native enzymes that could serve as potential surrogates for the aldehyde-forming acyl-CoA reductases and alcohol dehydrogenases present in organisms that synthesize higher-chain linear n-alcohols (Table 7). The product titer (0.33 g/L) and yield (8.3% w/w) achieved upon overexpression of YiaY were higher than previously reported (Table 8).

(55) Synthesis of odd-chain n-alcohols was demonstrated by supplementing the medium with propionate as the precursor of propionyl-CoA (RCH.sub.3 in FIG. 1A). A clear shift in the distribution of n-alcohols was observed: odd-chain alcohols 1-pentanol, 1-heptanol, and 1-nonanol appeared as fermentation products and the synthesis of even-chain alcohols significantly decreased (FIG. 3d).

Example 4: Synthesis of Alkanes/Alkenes

(56) The CoA-thioester intermediates generated by the reversal of the -oxidation cycle can be converted to alkanes by a two-step pathway composed of an aldehyde-forming fatty-AcylCoA reductase and a fatty aldehyde decarbonylase (FIG. 5). Olefins, on the other hand, will also be synthesized from acyl-CoAs via a pathway that uses a head-to-head condensation mechanism followed by reduction and decarbonylation steps.

(57) Alkane-Biosynthesis Pathway:

(58) A two-step pathway will be used, which involves the reduction of acyl-CoA to fatty aldehydes by the action of fatty aldehyde-forming acyl-CoA reductases followed by the decarbonylation of the resulting aldehyde to alkane by aldehyde decarbonylases (FIG. 5). This pathway is different from a recently reported pathway for the synthesis of alkanes from fatty-acyl-ACP (acyl-acyl carrier protein). Our pathway uses an acyl-CoA reductase as opposed to the reported acyl-ACP reductase. We have already used native fatty aldehyde-forming acyl-CoA reductases in the synthesis of fatty alcohols. In addition, we will use heterologous fatty aldehyde-forming acyl-CoA reductases from Acinetobacter calcoaceticus (acr1) and Acinetobacter sp. strain M-1 (acrM) (Ishige et al., 2002).

(59) While both enzymes are active with a range of acyl-CoAs, the activity towards palmitoyl-CoA is very high: this is an important aspect because our strains engineered to produce fatty acids synthesize palmitic acid as the primary product (FIG. 3b), indicating the availability of palmitoyl-CoA for the fatty aldehyde-forming acyl-CoA reductases.

(60) For the second step of the pathway we will use an aldehyde decarbonylase from Synechococcus elongatus PCC7942 (PCC7942_orf1593) and other orthologs recently reported by Schirmer (2010).

(61) Genes encoding the aforementioned enzymes were clustered in the same expression vector (FIG. 6), which will be transformed into strains that we have already shown to be able to produce long-chain fatty acids from acyl-CoAs. Heterologous genes were codon-optimized for expression in E. coli. The effect of the expression levels of each enzyme in the pathway will be assessed through the use of different expression vectors, promoters and ribosomal binding sites, etc. Once alkane production has been verified, the vector carrying the alkane-biosynthesis pathway (FIG. 6) will be expressed in conjunction with a second vector carrying the -oxidation enzymes.

(62) The activity of proteins encoded by cloned genes will be quantified and the corresponding reactions characterized using in vitro analysis of enzyme kinetics and identification of reaction substrates and products using biochemical assays and NMR spectroscopy. Substrates with different chain length will be used in these assays.

(63) Olefin-Biosynthesis Pathway:

(64) The best-characterized pathway for the synthesis of olefins proceeds through a mechanism known as head-to-head condensation of acyl-CoAs and leads to the synthesis of long-chain olefins (C21-C31) with internal double bonds at the median carbon.

(65) The optimal functioning of this pathway will require the expression of the cluster of olefin-forming enzymes OleABCD from bacteria such as Xanthomonas campestris. Recent in vitro studies have shown that OleA catalyzes the condensation of fatty acyl groups in the first step of the pathway through a non-decarboxylative Claisen condensation mechanism. Purified OleA was shown to be active with fatty acyl-CoAs that ranged from C8 to C16 in length, with maximum activity towards palmitoyl-CoA. The other three genes encode a member of the /-hydrolase superfamily (OleB), a member of the AMPdependent ligase/synthase superfamily or acetyl-CoA synthetase-like superfamily (OleC), and a member of the short-chain dehydrogenase/reductase superfamily (OleD).

(66) The genes acr1, acrM, PCC7942_orf1593, oleABCD will be cloned in one expression vector and the effect of the expression levels of each enzyme in the pathway will be assessed through the use of different promoters and ribosomal binding sites, as described above. A second vector will be used to express the -oxidation enzymes. The vectors will be transformed into strains already shown to be able to produce long-chain fatty acids from acyl-CoAs. The activity of proteins encoded by the cloned genes will be quantified and the corresponding reactions characterized using in vitro analysis of enzyme kinetics and identification of reaction substrates and products using biochemical assays and NMR spectroscopy.

Example 5: Improving Efficiency of Reversed Cycle

(67) The synthesis and consumption of reducing equivalents is a key aspect for the efficient operation of the engineered pathway, we propose to improve its functioning by manipulating the enzymes responsible for trans-enoyl-CoA reduction and pyruvate oxidation (FIG. 1A and FIG. 5). This includes dissimilation of pyruvate through routes that preserve reducing equivalents (as opposed to releasing them in the form of hydrogen), use of NAD(P)H-dependent trans-enoyl-CoA reductases, and direct coupling between trans-enoyl-CoA reduction and pyruvate oxidation.

(68) Pyruvate can be converted to acetyl-CoA in E. coli through three main routes (FIG. 5): i) pyruvate formate-lyase (PFL), which generates formate as co-product, ii) pyruvate dehydrogenase (PDH), which generates CO.sub.2 and NADH, and iii) a YdbK, a predicted pyruvate:flavodoxin oxidoreductase, that also generates CO.sub.2 and transfer the electrons to the quinone pool. While the formate generated by PFL can be disproportionated to CO.sub.2 and hydrogen by the action of formate hydrogenlyase (FHL), this enzyme does not generate NAD(P)H.

(69) To address this issue, we will replace the native hydrogen-evolving FHL complex with an NAD-dependent formate dehydrogenase (FDH) from C. boidinii (FIG. 5). PDH and YdbK generate reducing equivalents in a form potentially usable by the engineered reversal of the b-oxidation pathway. However, effective functioning of PDH would require the use of anaerobic conditions or the replacement of the native PDH with an anaerobically active pyruvate dehydrogenase complex (PDH*) (Kim et al., 2007). In the case of YdbK, we propose the direct coupling of pyruvate oxidation and YdiO-catalyzed reduction of trans-enoyl-CoA. We will also evaluate the expression of a heterologous pyruvate-NADP oxidoreductase (PNO) from the mitochondrion of Euglena gracilis and from the apicomplexan Cryptosporidium parvum, which convert pyruvate to acetyl-CoA, CO.sub.2, and NADPH (Rotte et al., 2001).

(70) Two enzymes will be evaluated for the reduction of trans-enoyl-CoA, namely NAD(P)H-dependent trans-enoyl-CoA reductase from Euglena gracilis (Hoffmeister et al., 2005) and predicted E. coli acyl-CoA dehydrogenase (YdiO). In the case of YdiO, we have recently shown that this enzyme is required for the operation of the reversal of the b-oxidation pathway (Dellomonaco et al., 2011). The effect of availability of reducing equivalents in the form of NADPH or NADH will also be evaluated through manipulation of the flux through transhydrogenases as well as the carbon flux partitioning between Embden-Meyerhof-Parnas pathway, Pentose Phosphate pathway, and the Entner-Doudoroff pathway.

(71) Genes encoding some of the aforementioned enzymes have been cloned in appropriate expression vectors (FIG. 7 and FIG. 8) and will be transformed into strains able to produce alcohols, carboxylic acids, alkanes, and alkenes via a an engineered reversal of the -oxidation cycle. The encoded activities and corresponding reactions will be characterized using in vitro analysis of enzyme kinetics and identification of reaction substrates and products using biochemical assays and NMR spectroscopy.

(72) Conclusions:

(73) The functional reversal of the -oxidation cycle engineered in this work represents a new and highly efficient platform for the synthesis of advanced fuels and chemicals. Its superior nature is illustrated in the following balanced equation for the synthesis of n-alcohols from glucose: n/4 C.sub.6H.sub.12O.sub.6.fwdarw.C.sub.nH.sub.n+2O+n/2 CO.sub.2+(n/21)H.sub.2O+n/2 ATP, with n being the chain length of the n-alcohol (FIG. 1a). As can be seen, the engineered pathway has the potential to achieve the maximum yield of n-alcohols on glucose (66.7%, C-mole basis) and generates 1 ATP per each 2-C incorporated into the n-alcohol molecule. This ATP yield is equivalent to that of efficient homo-fermentative pathways found in nature such as ethanol and lactic acid fermentations. The high carbon and energy efficiency of the engineered reversal of the -oxidation cycle is possible because it uses acetyl-CoA directly as C2 donor during chain elongation (as opposed to first requiring ATP-dependent activation to malonyl-CoA) and it functions with acyl-CoA intermediates, which are the precursors of alcohols and other important products (FIG. 1a).

(74) The synthesis of n-alcohols through alternative metabolic routes, such as the fatty acid biosynthesis and keto-acid pathways, is less efficient. For example, the use of the fatty acid biosynthesis pathway results in the net consumption of 1 ATP per molecule of n-alcohol synthesized (FIG. 4). This inefficiency is due to the consumption of ATP in the synthesis of malonyl-ACP (reaction {circle around (7)} in FIG. 4), the C2 donor for chain elongation, and the use of acyl-ACP intermediates, which need to be converted to free acids and acylated (another ATP-consuming step) before their reduction to alcohols can be achieved (reactions {circle around (10)} and {circle around (11)} in FIG. 4). The recently proposed keto-acid pathway is also less efficient than the reversal of the -oxidation cycle: e.g. the maximum theoretical yield of n-hexanol, the highest-chain linear n-alcohol reported with the keto-acid pathway, is only 50% C-mole (2 Glucose.fwdarw.n-hexanol+ATP+2[H]+6 CO.sub.2).

(75) While the work reported here focused on the engineering of E. coli, the ubiquitous nature of -oxidation, aldehyde/alcohol dehydrogenase, and thioesterase enzymes will certainly enable the use of native metabolic engineering strategies to achieve the efficient synthesis of n-alcohols and fatty acids in other industrial organisms. A functional reversal of the -oxidation cycle also holds great promise for the combinatorial biosynthesis of a wide range of molecules of various chain lengths and functionalities. For example, thioesterases and aldehyde/alcohol dehydrogenases can also act on the other thioester intermediates of the engineered pathway to generate a host of products such as -keto acids and -keto alcohols, -hydroxy acids and 1,3-diols, and trans-.sup.2-fatty acids and trans-.sup.2-alcohols (FIG. 1a), as well as alkanes and alkenes (FIG. 5).

(76) TABLE-US-00002 TABLE 1 Functional characterization of the engineered reversal of the -oxidation cycle during the synthesis of n-butanol Table 1a. Activities of -oxidation and butanol dehydrogenase enzymes in wild-type and engineered strains Enzyme activity (mol/mg protein/min) standard deviation Strain THL.sup.a HBD.sup.a CRT.sup.a BDH.sup.a MG1655 n.d. 0.002 0.000 n.d. 0.014 0.001 RB02.sup.b 0.310 0.079 0.304 0.032 0.339 0.049 0.004 0.002 RB02 [yqeF+ fucO+] 0.498 0.036 0.292 0.013 0.334 0.017 0.298 0.020 Table 1b. Butanol synthesis, glucose utilization, and cell growth in strain RB02 and its derivatives.sup.c Butanol produced Glucose utilized Cell growth Strain.sup.d Yield (g/g) Concentration (g/L) (g/L) (g/L) RB02 [yqeF+ fucO+] 0.182 1.85 10.19 0.72 Reaction {circle around (1)}: yqeF (predicted acyltransferase) RB02 yqeF [fucO+] 0.019 0.10 5.19 0.32 RB02 [fucO+] 0.063 0.23 3.66 0.41 RB02 yqeF [fucO+ 0.159 1.11 7.00 0.43 yqeF+] Reactions {circle around (2)} and {circle around (3)}: fadB (3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase) RB02 fadB [yqeF+ 0.000 0.00 2.96 0.19 fucO+] RB02 fadB [yqeF+ 0.157 0.16 5.86 0.52 fadB+] RB02 fadB [yqeF+] 0.000 0.00 1.84 0.11 Reaction {circle around (4)}: ydiO (predicted acyl-CoA dehydrogenase) RB02 ydiO [yqeF+ 0.038 0.04 3.14 0.18 fucO+] RB02 ydiO [yqeF+ 0.163 0.16 5.94 0.57 ydiO+] RB02 ydiO [yqeF+] 0.018 0.02 1.66 0.13 Reaction {circle around (5)}: fucO (L-1,2-propanediol oxidoreductase/n-butanol dehydrogenase) RB02 fucO [yqeF+] 0.040 0.04 2.75 0.38 RB02 fucO [yqeF+ 0.151 0.11 5.64 0.51 fucO+] RB02 [yqeF+] 0.088 0.35 4.00 0.68 .sup.aTHL: thiolase; HBD: hydroxy-acyl-CoA dehydrogenase; CRT: crotonase; BDH: butanol dehydrogenase; n.d.: not detected. .sup.bThe genotype of strain RB02 is as follows: fadR atoC(c) crp* arcA ptaadhEfrdA. .sup.cExperiments were run for 24 hours in shake flasks using glucose (1% w/v) minimal medium. .sup.dStrains were grouped based on the relevance of their genotypes for specified reactions (see FIG. 1a).

(77) TABLE-US-00003 TABLE 2 Cell growth, glucose utilization, product synthesis, and carbon recovery for wild- type and engineered strains grown on glucose minimal medium Table 2A. n-butanol synthesis in wild-type and engineered E. coli strains Concentration (g/L) Glucose Strain Cells utilized Butanol % C-recovery.sup.b MG1655 0.85 7.33 0.00 88.77 fadR atoC(c) 0.71 5.16 0.00 85.78 fadR atoC(c) arcA crp crp* (RB01) 0.53 6.43 0.00 90.23 RB01 adhE frdA pta (RB02) 0.29 1.76 0.00 94.09 RB02 [yqhD+] 0.49 4.27 0.11 91.70 RB02 [fucO+] 0.41 3.66 0.23 98.48 RB02 [yqeF+] 0.68 4.00 0.35 97.13 RB02 [yqeF+ fucO+] 0.72 10.19 1.85 86.05 RB02 yqhD [yqeF+ fucO+] 0.49 7.89 1.90 84.15 RB02 yqhD eutE [yqeF+ fucO+] 0.66 7.66 2.15 93.54 Table 2B. Synthesis of higher chain (C > 4) n-alcohols by derivatives of strain RB03 (RB02 yqhD fucO) Concentration (g/L) Glucose % C- Strain Cells utilized n-C6OH n-C8OH n-C10OH recovery.sup.b RB03 fadD [fadBA+] 0.71 5.27 0.000 0.000 0.000 90.38 RB03 [fadBA+ yiaY+] 0.87 7.19 0.081 0.035 0.130 72.71 RB03 fadD [fadBA+ 0.73 5.06 0.170 0.080 0.170 91.97 yiaY+] RB03 [fadBA+ eutG+] 0.73 4.68 0.170 0.040 0.000 97.50 RB03 fadD [fadBA+ 0.52 4.00 0.170 0.070 0.010 90.36 eutG+] RB03 [fadBA+ betA+] 0.70 4.06 0.180 0.000 0.000 90.27 RB03 fadD [fadBA+ 0.72 3.98 0.210 0.100 0.020 91.16 betA+] Table 2C. Synthesis of long-chain (C > 10) saturated fatty acids by RB03 derivatives Concentration (g/L) Glucose Strain Cells utilized C10:0 C12:0 C14:0 C16:0 C18:0 % C-recovery.sup.b RB03 fadD 1.02 6.13 0.000 0.000 0.000 0.000 0.000 97.28 [fadBA+] RB03 fadD 0.72 3.07 0.000 0.000 0.000 0.700 0.180 98.14 [fadBA+ fadM+] RB03 fadD 0.53 2.87 0.050 0.000 0.080 0.450 0.100 91.94 [fadBA+ yciA+] .sup.aData represent averages from three samples taken from shake flask cultures grown on 2% (w/v) glucose minimal medium for: A. 24 h, B. 48 h, and C. 72 h. .sup.bCarbon recovery was calculated by multiplying the moles of product per mole of glucose times the number of carbon atoms in the molecule.

(78) TABLE-US-00004 TABLE 3 Strains used in this study Strains Description/Genotype Source MG1655 F--ilvG-rfb-50 rph-1 Cronon fadR atoC(c) MG1655 fadR atoC(con) Dellomonoco RB01 MG1655 fadR atoC(con) arcA crp::crp* This study RB02 MG1655 fadR atoC(con) arcA crp::crp* adhE pta frdA This study RB02 yqhD MG1655 fadR atoC(con) arcA crp::crp* adhE pta frdA yqhD This study RB02 eutE MG1655 fadR atoC(con) arcA crp::crp* adhE pta frdA eutE yqhD This study yqhD RB02 yqeF MG1655 fadR atoC(con) arcA crp::crp* adhE pta frdA yqeF This study R802 fadB MG1655 fadR atoC(con) arcA crp::crp* adhE pta frdA fadB This study RB02 ydiO MG1655 fadR atoC(con) arcA crp::crp* adhE pta frdA ydiO This study RB02 fucO MG1655 fadR atoC(con) arcA crp::crp* adhE pta frdA fucO This study RB03 MG1655 fadR atoC(con) arcA crp::crp* adhE pta frdA fucO yqhD This study RB03 fadD MG1655 fadR atoC(con) arcA crp::crp* adhE pta frdA fucO yqhD fadD This study

(79) TABLE-US-00005 TABLE 4 Plasmids used in this study Plasmid Description/Genotype Source pTrcHis2A pTrcHis2A (pBR322-derived), oriR pMB1, lacI.sup.q, bla Invitrogen (Carlsbad, CA) pTH fadBA E. coli fadBA genes under trc promoter and lacI.sup.q This study control in pTrcHis2A pTH yqeF E. coli yqeF gene under trc promoter and lacI.sup.q control This study in pTrcHis2A pZS blank oriR pSC101*, tetR, cat, contains P.sub.LtetO-1 Yazdani pZS atoB E. coli atoB gene under control of P.sub.LtetO-1(tetR, oriR This study SC101*, cat) pZS betA E. coli betA gene under control of P.sub.LtetO-1(tetR, oriR This study SC101*, cat) pZS eutG E. coli eutG gene under control of P.sub.LtetO-1(tetR, oriR This study SC101*, cat) pZS fadB E. coli fadB gene under control of P.sub.LtetO-1(tetR, oriR This study SC101*, cat) pZS fadM E. coli fadM gene under control of P.sub.LtetO-1(tetR, oriR This study SC101*, cat) pZS fucO E. coli fucO gene under control of P.sub.LtetO-1(tetR, oriR This study SC101*, cat) pZS yciA E. coli yciA gene under control of P.sub.LtetO-1(tetR, oriR This study SC101*, cat) pZS ydiO E. coli ydiO gene under control of P.sub.LtetO-1(tetR, oriR This study SC101*, cat) pZS yiaY E. coli yiaY gene under control of P.sub.LtetO-1(tetR, oriR This study SC101*, cat) pZS yqhD E. coli yqhD gene under control of P.sub.LtetO-1(tetR, oriR This study SC101*, cat)

(80) TABLE-US-00006 TABLE 5 Comparison of crp, fadR, and atoSC loci of engineered strain RB02 (fadR atoC(c) arcA crp::crp* adhE pta frdA) and wild-type MG1655. Gene Accession # for Mutations/insertions locus RB02 sequence in RB02 sequence Comments crp BankIt1445305 I113L, T128I, A145T Mutations collectively reduce dependence on cAMP for activation of Seq1 JF781281 catabolic genes by CRP, as previously described.sup.12.45. fadR BankIt1446148 IS5 insertion between bp Inactivation of fadR by IS5 insertion, which would preclude synthesis of Seq1 JF793627 395-396 of fadR gene C-terminal half of FadR and hence DNA binding. Characteristic phenotype of fadR inactivation previously confirmed in strain fadR atoC(c).sup.10. atoSC BankIt1445920 I129S atoC tranduced from LS5218 (constitutive atoC expression.sup.46). Seq1 JF793626 Characteristic phenotype of constitutive expression of ato operon confirmed in strain fadR atoC(c).sup.10.

(81) TABLE-US-00007 TABLE 6 Cell growth, glucose utilization, product synthesis, and carbon recovery for wild- type and engineered strains grown on glucose minimal medium.sup.a Table 6A. Synthesis of n-butanol in wild-type and engineered E. coli strains Concentration.sup.c (g/L) Strain.sup.b Cells Glucose utilized Butanol % C-recovery.sup.d MG1655 0.85 7.33 ND 88.77 fadR atoC(c) 0.71 5.16 ND 85.78 RB01 (fadR atoC(c) arcA crp*) 0.53 6.43 ND 90.23 RB02 (RB01 adhE frdA pta) 0.29 1.76 ND 94.09 RB02 [yqhD+] 0.49 4.27 0.11 91.70 RB02 [fucO+] 0.41 3.66 0.23 98.48 RB02 [yqeF+] 0.68 4.00 0.35 97.13 RB02 [yqeF+ fucO+] 0.72 10.19 1.85 86.05 MG1655 [yqeF+ fucO+] 0.94 7.95 ND 91.15 RB02 yqhD [yqeF+ fucO+] 0.49 7.89 1.90 84.15 RB02 yqhD eutE [yqeF+ fucO+] 0.66 7.66 2.15 93.54 Table 6B. Synthesis of 4-C carboxylic acids by derivatives of strain RB02 Concentration.sup.c (g/L) - trans-2- hydroxy- butenoic Strain.sup.b Cells Glucose utilized -keto-C4:0 C4:0 acid % C-recovery.sup.d RB02 0.29 1.76 ND ND ND 94.09 RB02 0.62 4.25 ND 0.032 0.010 95.30 [tesA+] RB02 0.57 4.30 0.024 ND ND 89.88 [tesB+] RB02 0.56 4.95 ND 0.045 0.012 91.08 [yqeF+ tesA+] RB02 0.42 3.34 ND 0.140 0.171 89.23 ydiO [yqeF+ tesA+] RB03 0.65 5.16 0.110 ND ND 97.17 [yqeF+ tesB+] RB03 0.62 5.03 0.450 ND ND 95.53 fadB [yqeF+ tesB+] Table 6C. Synthesis of long-chain (C > 10) saturated fatty acids by RB03 (RB02 yqhD fucO fadD) derivatives Concentration.sup.c (g/L) Extracellular FFAs (Free Fatty Acids) Glucose Total TotalFFAs/ % C- Strain.sup.b Cells utilized C10:0 C12:0 C14:0 C16:0 C18:0 FFAs.sup.e CDW recovery.sup.d MG1655 1.29 12.88 ND ND ND ND ND ND / 93.05 RB03 0.63 4.30 ND ND ND ND ND ND / 90.64 RB03 [fadBA+] 0.51 2.24 ND ND ND ND ND 0.090 0.175 91.55 RB03 [fadBA+ tesA+] 0.56 5.82 ND ND 0.110 ND ND .sup.f 92.02 RB03 [fadBA+ tesB+] 0.62 5.16 0.120 ND ND ND ND .sup.f 94.78 RB03 [fadBA+ yciA+] 0.53 2.87 0.050 ND 0.080 0.450 0.100 .sup.f 91.94 RB03 [fadBA+ fadM+] 0.72 3.07 ND ND ND 0.700 0.180 .sup.f 98.14 RB03 [fadM+].sup.g 0.55 3.30 ND ND 0.020 0.150 0.080 0.435 0.790 92.86 RB03 [fadBA.fadM+] 0.67 5.91 ND ND ND 0.740 0.500 1.370 2.035 97.19 MG1655 adhE pta frdA 0.38 5.04 ND ND ND ND ND ND / 86.44 fadD MG1655 adhE pta frdA 0.76 5.07 ND ND ND ND ND 0.070 0.092 94.15 fadD [fadM+] MG1655 adhE pta frdA 0.94 5.23 ND ND ND ND ND 0.261 0.279 96.17 fadD [fadM+].sup.g Table 6D. Synthesis of higher chain (C > 4) n-alcohols by derivatives of strain RB03 (RB02 yqhD fucO fadD) Concentration.sup.c (g/L) Glucose Strain.sup.b Cells utilized n-C6OH n-C8OH n-C10OH % C-recovery.sup.d MG1655 adhE pta frdA fadD [betA+] 0.65 4.16 ND ND ND 91.54 RB03 [fadBA+] 0.71 5.27 ND ND ND 90.38 RB03 [fadBA+ yiaY+] 0.73 5.06 0.170 0.080 0.170 91.97 RB03 [fadBA+ eutG+] 0.52 4.00 0.170 0.070 0.010 90.36 RB03 [fadBA+ betA+] 0.72 3.98 0.210 0.100 0.020 91.16 .sup.aData represent averages for three samples taken from shake flask cultures grown on 2% (w/v) glucose minimal medium. A. cultures were grown at 30 C. for 24 hours; B., C., D. cultures were grown at 37 C. for 48 hours. .sup.bAll genotypes are shown in Table 4. .sup.cND, not detectable. Minimum detection levels are: butanol, 5.84 mg l.sup.1; -keto-C4:0, 4.09 mg l.sup.1; -hydroxy-C4:0, 3.03 mg l.sup.1 trans-2-butenoic acid, 9.40 mg l.sup.1; C10:0, 21.76 mg l.sup.1; C12:0, 20.45 mg l.sup.1; C14:0, 27.12 mg l.sup.1; C16:0, 20.17 mg l.sup.1; C18:0, 16.42 mg l.sup.1; n-C6OH, 24.21 mg l.sup.1; n-C8OH, 26.41 mg l.sup.1; n-C10OH, 21.23 mg l.sup.1. .sup.dCarbon recovery was calculated as the ratio of total moles of carbon in products per moles of carbon in total glucose consumed and expressed on percentage basis. .sup.eFFAs, Free Fatty Acids .sup.fValues not measured .sup.gfadM was overexpressed from medium-copy vector pTrcHis2A (Invitrogen, Carlsbad, CA).

(82) TABLE-US-00008 TABLE 7 In silico identification of E. coli surrogates for higher-chain (C 4) aldehyde-forming acyl-CoA reductases and aldehyde/alcohol dehydrogenases (reaction in FIG. 1). Genes shown in bold were tested in this study. Surrogates identified via I-TASSER.sup.47 Source of gene sequences used to identify E. coli surrogates TM- EC- Organism Accession # Gene EC # FUNCTION EC # Score.sup.a Score.sup.b Gene Identification of E. coli surrogates for higher-chain (C 4) fatty aldehyde-forming acyl-CoA reductase Clostridium P13604 adh1 1.1.1. NADH-dependent 1.1.1.77 0.9404 2.2424 fucO saccharobutylicum.sup.49 butyraldehyde/butanol dehydrogenase 1.1.1.1. 0.8679 1.8069 yiaY adhE adhP frmA 1.1.1.6 0.7979 1.4449 gldA Pseudomonas sp. strain Q52060 dmpF 1.2.1.10 Acetaldehyde 1.2.1.12 0.7439 1.3969 gapA CF600.sup.50 dehydrogenase Acinetobacter sp. Q8RR58 acrM 1.2.1.50 Acyl coenzyme A <1.1.sup.b Strain M-1.sup.51 reductase Acinetobacter P94129 acr1 1.2.1.n2 Fatty acyl-CoA <1.1.sup.b calcoaceticus.sup.52 reductase Identification of E. coli surrogates for higher-chain (C 4) aldehyde/alcohol dehydrogenases Geobacillus A4IP64 GTNG_1754 1.1.1. Alcohol 1.1.1.202 0.9565 2.5069 yqhD thermodenitrificans.sup.51 Dehydrogenase 1.1.1.77 0.9312 2.1532 fucO 1.1.1.1. 0.8512 1.9177 yiaY adhE adhP frmA 1.1.1.6 0.7715 1.5259 gldA Pseudomonas Q00593 alkJ 1.1.99. Alcohol oleovorans.sup.54 Dehydrogenase Thermococcus sp. ESI.sup.55 CIIWT4 adh 1.1.1.1. Iron alcohol 1.1.1.202 0.9301 2.2696 yqhD dehydrogenase 1.1.1.77 0.9041 1.9642 fucO 1.1.1.1. 0.8352 1.8286 yiaY adhE adhP frmA 1.1.1.6 0.7800 1.3793 gldA Thermococcus Y14015 1.1.1. Alcohol hydrothermalis.sup.56 Dehydrogenase Sulfolobus tokodaii.sup.57 Q976Y8 ST0053 Hypothetical alcohol 1.1.1.1. 0.9655 1.8286 yiaY dehydrogenase adhE adhP frmA Surrogates identified via protein BLAST.sup.48 E- Organism EC # Gene value.sup.c Identity Similarity Identification of E. coli surrogates for higher-chain (C 4) fatty aldehyde-forming acyl-CoA reductase Clostridium 1.1.1.1 adhE 2.0E91 42% 62% saccharobutylicum.sup.49 1.1.1.77 fucO 5.0E66 35% 58% 1.1.1. yiaY 2.0E65 37% 56% 1.1.. eutG 4.0E61 35% 54% 1.1.1. yqhD 6.0E28 25% 46% Pseudomonas sp. strain 1.2.1.10 mhpF 6.0E146 79% 92% CF600.sup.50 Acinetobacter sp. 1... ucpA 5.0E20 31% 49% Strain M-1.sup.51 1... ybbO 4.0E19 30% 50% 1.1.1. ydfG 6.0E18 27% 47% 1.1.1.69 idnO 9.0E18 29% 49% 1.1.1.100 fabG 9.0E17 31% 51% Acinetobacter 1.1.1.100 fabG 2.0E18 31% 53% calcoaceticus.sup.52 1.1.1.69 idnO 3.0E17 29% 48% 1.1.1. ydfG 1.0E16 27% 47% 1... ybbO 1.0E16 28% 44% 1... ucpA 2.0E16 31% 52% Identification of E. coli surrogates for higher-chain (C 4) aldehyde/alcohol dehydrogenases Geobacillus 1.1.. eutG 3.0E57 39% 57% thermodenitrificans.sup.51 1.1.1.1 yiaY 3.0E54 33% 52% 1.1.1.77 fucO 2.0E52 34% 53% 1.1.1.1. adhE 3.0E43 34% 52% 1.1.1.1 yqhD 1.0E18 28% 46% Pseudomonas 1.1.99.1 betA 2.0E104 40% 59% oleovorans.sup.54 Thermococcus sp. ESI.sup.55 1.1.1.1 yiaY 7.0E40 33% 50% 1.1.1.1 yqhD 4.0E30 30% 46% 1.1.. eutG 3.0E27 30% 46% 1.1.1.1. adhE 9.0E27 29% 48% Thermococcus 1.1.1.1 yiaY 4.0E37 31% 48% hydrothermalis.sup.56 1.1.. eutG 2.0E34 31% 47% 1.1.1.1. adhE 1.0E30 30% 50% 1.1.1.77 fucO 4.0E30 30% 45% 1.1.1.1 yqhD 5.0E19 30% 44% Sulfolobus tokodaii.sup.57 1.1.1.1. adhP 4.0E33 31% 50% 1... ydjJ 8.0E26 30% 50% 1... yphC 2.0E21 31% 47% 1... yahK 2.0E21 29% 45% 1.1.1. rspB 6.0E21 29% 49% 1.1.1.103 tdH 9.0E20 27% 47% 1... yjjN 3.0E19 26% 45% 1.1.. gatD 1.0E18 28% 44% .sup.aThe Template Modeling-score (TM-score) is defined to assess the topological similarity of protein structure pairs. Its value ranges between 0 and 1, and a higher score indicates better structural match. Statistically, a TM-score <0.17 means a randomly selected protein pair with the gapless alignment taken from PDB.sup.47. .sup.bAn EC-score >1.1 is a good indicator of the functional similarity between the query and the identified enzyme analogs.sup.47. .sup.cThe BLAST E-value measures the statistical significance threshold for reporting protein sequence matches against the organism genome database; the default threshold value is 1E5, in which 1E5 matches would be expected to occur by chance, according to the stochastic model of Karlin and Altschul (http://www.ncbi.nlm.nih.gov/BLAST/tutorial/).

(83) TABLE-US-00009 TABLE 8A Summary of organisms that have been engineered to produce higher-chain (C 4) linear n-alcohols and long-chain (C 10) fatty acids Engineered Titer (g/L)/ Fermentation Carbon Medium/ Product Host Yield (% w/w).sup.a Time (hr) Source Cultivation Reference Higher-chain (C 4) linear n-alcohols n-butanol (C4) E. coli 0.55/2.8 24 Glycerol Rich/Batch 35 E. coli 0.82/3.3 100 Glucose Rich/Batch 7.8 E. coli 1.2/6.1 60 Glucose Rich-MM/HCD.sup.b 58 E. coli 0.58/11.6 48 Glycerol Rich/Batch 59 S. cerevisiae 0.002/0.0001 72 Galactose Rich/Batch 60 B. subtilis 0.024/0.48 72 Glycerol Rich/Batch 59 P. putida 0.120/2.4 72 Glycerol Rich/Batch 59 L. lactis 0.028/ Glucose Rich/Batch 61 L. buchneri 0.066/ Glucose Rich/Batch 61 L. brevis 0.3/1.5 60 Glucose Rich/Batch 41 E. coli 2.05/13.4 96 Palmitic Acid MM/Fed-Batch 10 E. coli 4.65/28 72 Glucose Rich/Batch 9 E. coli 14.5/32.8 36 Glucose MM/Batch This work n-hexanol (C6) E. coli 0.04/0.2 40 Glucose Rich/Batch 29 Fatty alcohols E. coli 0.06/0.3 Glucose MM/Batch 2 (Distribution of C10, C12, C14, and C16) Higher-chain n- E. coli 0.42/8.3 48 Glucose MM/Batch This work alcohols (C6 to C10)

(84) TABLE-US-00010 TABLE 8B Summary of organisms that have been engineered to produce higher-chain (C 4) linear n-alcohols and long-chain (C 10) fatty acids Engineered Titer (g/L)/ Fermentation Carbon Medium/ Host Yield (% w/w).sup.a Time (hr) Source Cultivation Reference Product Long-chain (C 10) fatty acids Fatty Acids E. coli 1.2/6 Glucose MM/Batch 2.sup.c (Predominantly C14) Fatty Acids E. coli 2.5/ 22 Glycerol MM/Fed- 62.sup.c (Wide Distribution) Batch/HCD Fatty Acids E. coli 0.81/16 29 Glycerol Rich/Batch 4.sup.c (Predominantly C12) Fatty Acids E. coli 6.6/28 60 Glucose MM/Batch This work (Predominantly C16, C18) .sup.aFor products with carbon length distributions, titer represents the sum of products of all chain length produced. Yield assumes all the carbon source was consumed when carbon source consumption data not given in reference. .sup.bTwo-phase, high cell density (HCD) culture grown first in rich medium and then incubated in minimal medium (MM). .sup.cTiters reported refer to total (i.e. sum of intracellular and extracellular) free fatty acids.

(85) TABLE-US-00011 TABLE 9 Homology analysis and functional annotation of E. coli ydi genes Current annotation.sup.a Functional homologues identified Gene Sequence-based homologues identified via protein BLAST.sup.48 via I-TASSER.sup.47 name Function Gene Function E-value.sup.b Coverage Similarity Function TM-Score.sup.c EC-Score.sup.d ydiO Predicted caiA Crotonobetainyl-CoA 1.0E102 99% 65% Butyryl-CoA 0.9584 2.2964 acyl-CoA reductase dehydrogenase dehydrogenase aidB Isovaleryl-CoA 2.0E14 64% 47% Acyl-CoA 0.9618 2.1730 dehydrogenase dehydrogenase fadE Acyl-CoA dehydrogenase 0.001 81% 37% ydiQ Putative electron fixA probable flavoprotein subunit 3.0E68 99% 71% Adenosine kinase <1.1 transfer required for anaerobic flavoprotein carnitine metabolism subunit ydiR Putative electron fixB probable flavoprotein subunit 6.0E75 100% 64% transfer required for anaerobic flavoprotein carnitine metabolism subunit ydiS Predicted fixC flavoprotein (electron 5.0E152 100% 78% Electron-transferring- 0.9404 1.8378 oxidoreductase transport) flavoprotein with dehydrogenase FAD/NAD(P)- vgcN predicted oxidoreductase with 9.0E93 99% 62% binding FAD/NAD(P)-binding domain domain ydiT Ferredoxin-like fixX putative ferredoxin possibly 3.0E33 96% 62% Electron-transferring- 0.9006 1.5362 protein involved in anaerobic flavoprotein carnitine metabolism dehydrogenase ygcO predicted 4Fe4S cluster- 1.0E18 89% 62% containing protein .sup.aAs annotated in Ecocyc.sup.63. Also reported by Campbell, J. W. and coworkers.sup.64. .sup.bThe BLAST E-value measures the statistical significance threshold for reporting protein sequence matches against the organism genome database; the default threshold value is 1E5, in which 1E5 matches would be expected to occur by chance, according to the stochastic model of Karlin and Altschul ncbi.nlm.nih.gov/BLAST/tutorial/). .sup.cThe Template Modeling-score (TM-score) is defined to assess the topological similarity of protein structure pairs. Its value ranges between 0 and 1, and a higher score indicates better structural match. Statistically, a TM-score <0.17 means a randomly selected protein pair with the gapless alignment taken from PDB.sup.47. .sup.dAn EC-score >1.1 is a good indicator of the functional similarity between the query and the identified enzyme analogs.sup.47.

(86) TABLE-US-00012 TABLE 10 Thermodynamic analysis of the engineered reversal of the -oxidation cycle Standard G, Reaction number and enzyme name (gene) (Min G.sub.r, Max G.sub.r) [kcal/mol].sup.a {circle around (1)} Thiolase (yqeF, fadA) 7.1 2 Acetyl-CoA .fwdarw. Acetoacetyl-CoA + CoA-SH (1.9, 16.1) {circle around (2)} Hydroxyacyl-CoA dehydrogenase (fadB) 3.7 Acetoacetyl-CoA + NADH + H.sup.+ .fwdarw. 3-hydroxybutyryl-CoA + NAD.sup.+ (12.7, 5.3) {circle around (3)} Enoyl-CoA hydratase (fadB) 2.1 3-hydroxybutyryl-CoA .fwdarw. Crotonyl-CoA + H.sub.2O (2.4, 6.6) {circle around (4)} Acyl-CoA dehydrogenase (coupled to ubiquinone, fadE).sup.b 5.7 Crotonyl-CoA + UQH.sub.2 .fwdarw. Butyryl-CoA + UQ (3.3, 14.7) {circle around (4)} Enoyl-CoA reductase (coupled to ferredoxin, ydiO-ydiQRST).sup.b 16.5 Crotonyl-CoA + Fd.sup.2 .fwdarw. Butyryl-CoA + Fd (25.5, 7.5) Operation of -oxidation reversal coupled to ubiquinone 11.2 Operation of -oxidation reversal coupled to ferredoxin 11.0 .sup.aStandard G of formation values estimated using the group contribution method.sup.65 and used to calculate the standard G of reaction.sup.66. Minimum and maximum G, values calculated assuming standard conditions (298.15 K, pH 7) with minimum and maximum metabolite concentrations set to 0.00001M and 0.02M, respectively.sup.66. Listed G.sub.r values are in good agreement with experimentally measured/calculated G, values.sup.67,68. .sup.bCalculation of G.sub.r for enoyl-CoA reductase used standard reduction potentials from Thauer et al.sup.69.

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