The ald Gene, Encoding a Coenzyme A-Acylating Aldehyde Dehydrogenase, Distinguishes Clostridium beijerinckii and Two Other Solvent-Producing Clostridia from Clostridium acetobutylicum

The coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH) catalyzes a key reaction in the acetone- and butanol (solvent)-producing clostridia. It reduces acetyl-CoA and butyryl-CoA to the corresponding aldehydes, which are then reduced by alcohol dehydrogenase (ADH) to form ethanol and 1-butanol. The ALDH of Clostridium beijerinckii NRRL B593 was purified. It had no ADH activity, was NAD(H) specific, and was more active with butyraldehyde than with acetaldehyde. The N-terminal amino acid sequence of the purified ALDH was determined. The open reading frame preceding the ctfA gene (encoding a subunit of the solvent-forming CoA transferase) of C. beijerinckii NRRL B593 was identified as the structural gene (ald) for the ALDH. The ald gene encodes a polypeptide of 468 amino acid residues with a calculated M_r of 51,353. The position of the ald gene in C. beijerinckii NRRL B593 corresponded to that of the aad/adhE gene (encoding an aldehyde-alcohol dehydrogenase) of Clostridium acetobutylicum ATCC 824 and DSM 792. In Southern analyses, a probe derived from the C. acetobutylicum aad/adhE gene did not hybridize to restriction fragments of the genomic DNAs of C. beijerinckii and two other species of solvent-producing clostridia. In contrast, a probe derived from the C. beijerinckii ald gene hybridized to restriction fragments of the genomic DNA of three solvent-producing species but not to those of C. acetobutylicum, indicating a key difference among the solvent-producing clostridia. The amino acid sequence of the ALDH of C. beijerinckii NRRL B593 was most similar (41% identity) to those of the eutE gene products (CoA-acylating ALDHs) of Salmonella typhimurium and Escherichia coli, whereas it was about 26% identical to the ALDH domain of the aldehyde-alcohol dehydrogenases of C. acetobutylicum, E. coli, Lactococcus lactis, and amitochondriate protozoa. The predicted secondary structure of the C. beijerinckii ALDH suggests the presence of an atypical Rossmann fold for NAD⁺ binding. A comparison of the proposed catalytic pockets of the CoA-dependent and CoA-independent ALDHs identified 6 amino acids that may contribute to interaction with CoA.     

Alcohol dehydrogenase: multiplicity and relatedness in the solvent-producing clostridia

Abstract: Alcohol dehydrogenase (ADH) is a key enzyme for the production of butanol, ethanol, and isopropanol by the solvent-producing clostridia. Initial studies of ADH in extracts of several strains of Clostridium acetobutylicum and C. beijerinckii gave conflicting molecular properties. A more coherent picture has emerged because of the following results: (i) identification of ADHs with different coenzyme specificities in these species; (ii) discovery of structurally conserved ADHs (type 3) in three solvent-producing species; (iii) isolation of mutants with deficiencies in butanol production and restoration of butanol production with a cloned alcohol/aldehyde dehydrogenase gene; and (iv) resolution of various ' C. acetobutylicum ' cultures into four species. The three ADH isozymes of C. beijerinckii NRRL B592 have high sequence similarities to ADH-1 of Clostridium sp. NCP 262 (formerly C. acetobutylicum P262) and to the ADH domain of the alcohol/aldehyde dehydrogenase of C. acetobutylicum ATCC 824/DSM 792. The NADH-dependent activity of the ADHs from C. beijerinckii NRRL B592 and the BDHs from C. acetobutylicum ATCC 824 is profoundly affected by the pH of the assay, and the relative importance of NADH and NADPH to butanol production may be misappraised when NAD(P)H-dependent activities were measured at different pH values. The primary/secondary ADH of isopropanol-producing C. beijerinckii is a type-1 enzyme and is highly conserved in Thermoanaerobacter brockii (formerly Thermoanaerobium brockii ) and Entamoeba histolytica . Several solvent-forming enzymes (primary ADH, aldehyde dehydrogenase, and 3-hydroxybutyryl-CoA dehydrogenase) are very similar between C. beijerinckii and the species represented by Clostridium sp. NCP 262 and NRRL B643. The realization of such relationships will facilitate the elucidation of the roles of different ADHs because each type of ADH can now be studied in an organism most amenable to experimental manipulations.     

Coenzyme A-acylating aldehyde dehydrogenase from Clostridium beijerinckii NRRL B592

Acetaldehyde and butyraldehyde are substrates for alcohol dehydrogenase in the production of ethanol and 1-butanol by solvent-producing clostridia. A coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH), which also converts acyl-CoA to aldehyde and CoA, has been purified under anaerobic conditions from Clostridium beijerinckii NRRL B592. The ALDH showed a native molecular weight (Mr) of 100,000 and a subunit Mr of 55,000, suggesting that ALDH is dimeric. Purified ALDH contained no alcohol dehydrogenase activity. Activities measured with acetaldehyde and butyraldehyde as alternative substrates were copurified, indicating that the same ALDH can catalyze the formation of both aldehydes for ethanol and butanol production. Based on the Km and Vmax values for acetyl-CoA and butyryl-CoA, ALDH was more effective for the production of butyraldehyde than for acetaldehyde. ALDH could use either NAD(H) or NADP(H) as the coenzyme, but the Km for NAD(H) was much lower than that for NADP(H). Kinetic data suggest a ping-pong mechanism for the reaction. ALDH was more stable in Tris buffer than in phosphate buffer. The apparent optimum pH was between 6.5 and 7 for the forward reaction (the physiological direction; aldehyde forming), and it was 9.5 or higher for the reverse reaction (acyl-CoA forming). The ratio of NAD(H)/NADP(H)-linked activities increased with decreasing pH. ALDH was O2 sensitive, but it could be protected against O2 inactivation by dithiothreitol. The O2-inactivated enzyme could be reactivated by incubating the enzyme with CoA in the presence or absence of dithiothreitol prior to assay.     

Coenzyme A-acylating aldehyde dehydrogenase from Clostridium beijerinckii NRRL B592.

Acetaldehyde and butyraldehyde are substrates for alcohol dehydrogenase in the production of ethanol and 1-butanol by solvent-producing clostridia. A coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH), which also converts acyl-CoA to aldehyde and CoA, has been purified under anaerobic conditions from Clostridium beijerinckii NRRL B592. The ALDH showed a native molecular weight (Mr) of 100,000 and a subunit Mr of 55,000, suggesting that ALDH is dimeric. Purified ALDH contained no alcohol dehydrogenase activity. Activities measured with acetaldehyde and butyraldehyde as alternative substrates were copurified, indicating that the same ALDH can catalyze the formation of both aldehydes for ethanol and butanol production. Based on the Km and Vmax values for acetyl-CoA and butyryl-CoA, ALDH was more effective for the production of butyraldehyde than for acetaldehyde. ALDH could use either NAD(H) or NADP(H) as the coenzyme, but the Km for NAD(H) was much lower than that for NADP(H). Kinetic data suggest a ping-pong mechanism for the reaction. ALDH was more stable in Tris buffer than in phosphate buffer. The apparent optimum pH was between 6.5 and 7 for the forward reaction (the physiological direction; aldehyde forming), and it was 9.5 or higher for the reverse reaction (acyl-CoA forming). The ratio of NAD(H)/NADP(H)-linked activities increased with decreasing pH. ALDH was O2 sensitive, but it could be protected against O2 inactivation by dithiothreitol. The O2-inactivated enzyme could be reactivated by incubating the enzyme with CoA in the presence or absence of dithiothreitol prior to assay.     

Butanol-Ethanol Dehydrogenase and Butanol-Ethanol-Isopropanol Dehydrogenase: Different Alcohol Dehydrogenases in Two Strains of Clostridium beijerinckii (Clostridium butylicum)

Alcohol-producing strains of Clostridium beijerinckii (Clostridium butylicum) produce, besides acetone, either n-butanol and ethanol or n-butanol, ethanol, and isopropanol as their characteristic products. Alcohol dehydrogenase has been isolated from a strain (NRRL B593) of C. beijerinckii producing isopropanol and from a strain (NRRL B592) not producing isopropanol. Butanol-ethanol dehydrogenase activities were present in both strains, but isopropanol dehydrogenase activity was present only in the isopropanol-producing strain. The butanol-ethanol dehydrogenase of strain NRRL B592 had M(r) 66,000 and a K(m) of 6 muM for butyraldehyde. In contrast, the butanol-ethanol-isopropanol dehydrogenase of strain NRRL B593 had a M(r) 100,000 and K(m)s of 9.5 and 1.0 mM for butyraldehyde and acetone, respectively. In a purification by four different types of separatory methods (DEAE-cellulose, hydroxyapatite, Sephacryl S-300, and Matrex Gel Red A), butanol-ethanol-isopropanol dehydrogenase activities of strain NRRL B593 were purified up to 200-fold (10 to 30% yield), and these activities were not separated. Gel electrophoresis followed by activity stain also revealed distinct mobilities for the butanol-ethanol dehydrogenase of strain NRRL B592 and the butanol-ethanol-isopropanol dehydrogenase of strain NRRL B593. In cell extracts from both strains, a higher alcohol dehydrogenase activity was measured with NADP(H) than with NAD(H). The 150- to 200-fold-purified alcohol dehydrogenase from strain NRRL B593 did not show any NAD(H)-linked activities. The K(m) for NADPH was 31 muM (with butyraldehyde as cosubstrate) and 18 muM (with acetone as cosubstrate) for the alcohol dehydrogenase of strain NRRL B593. This study showed that the alcohol dehydrogenases from two strains of C. beijerinckii differed significantly.     

Purification and properties of 3-hydroxybutyryl-coenzyme A dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593.

The enzyme 3-hydroxybutyryl-coenzyme A (CoA) dehydrogenase has been purified 45-fold to apparent homogeneity from the solvent-producing anaerobe Clostridium beijerinckii NRRL B593. The identities of 34 of the N-terminal 35 amino acid residues have been determined. The enzyme exhibited a native M(r) of 213,000 and a subunit M(r) of 30,800. It is specific for the (S)-enantiomer of 3-hydroxybutyryl-CoA. Michaelis constants for NADH and acetoacetyl-CoA were 8.6 and 14 microM, respectively. The maximum velocity of the enzyme was 540 mumol min-1 mg-1 for the reduction of acetoacetyl-CoA with NADH. The enzyme could use either NAD(H) or NADP(H) as a cosubstrate; however, kcat/Km for the NADH-linked reaction was much higher than the apparent value for the NADPH-linked reaction. Also, NAD(H)-linked activity was less sensitive to changes in pH than NADP(H)-linked activity was. In the presence of 9.5 microM NADH, the enzyme was inhibited by acetoacetyl-CoA at concentrations as low as 20 microM, but the inhibition was relieved as the concentration of NADH was increased, suggesting a possible mechanism for modulating the energy efficiency during growth.     

Metabolic engineering of Clostridium acetobutylicum ATCC 824 for isopropanol-butanol-ethanol fermentation

Clostridium acetobutylicum naturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineered C. acetobutylicum can be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B-593 (i.e., adh(B-593)) in C. acetobutylicum ATCC 824. To increase the total alcohol titer, a synthetic acetone operon (act operon; adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in the buk gene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged by in situ removal of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.     

Purification and properties of an acetoacetyl coenzyme A-reacting phosphotransbutyrylase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593

During the study of acetoacetyl coenzyme A (CoA)-reacting enzymes of Clostridium beijerinckii NRRL B593, a phosphate-dependent acetoacetyl-CoA-utilizing activity was detected in protein fractions devoid of thiolase and phosphotransacetylase. Further purification of this acetoacetyl-CoA-utilizing activity yielded an enzyme which may be designated as phosphotransbutyrylase (PTB; phosphate butyryltransferase [EC 2.3.1.19]). PTB from C. beijerinckii NRRL B593 was purified 160-fold with a yield of 14% and, with the best fractions, purified 190-fold to near homogeneity. It showed a native Mr of 205,000 and a subunit Mr of 33,000. PTB activity was sensitive to pH changes within the physiological range of 6 to 8. PTB exhibited a broad substrate specificity. The Km values at pH 7.5 for butyryl-CoA, acetoacetyl-CoA, and acetyl-CoA were 0.04, 1.10, and 3.33 mM, respectively. The Vmax values with butyryl-CoA and acetoacetyl-CoA were comparable, but the Vmax/Km was higher for butyryl-CoA than for acetoacetyl-CoA. An apparent Km of 6.5 mM for phosphate was obtained with butyryl-CoA as the cosubstrate, whereas it was 12.9 mM with acetoacetyl-CoA as the cosubstrate. It remains to be established whether the putative compound acetoacetyl phosphate is produced in the PTB-catalyzed reaction with acetoacetyl-CoA.     

Engineered Synthetic Pathway for Isopropanol Production in Escherichia coli

A synthetic pathway was engineered in Escherichia coli to produce isopropanol by expressing various combinations of genes from Clostridium acetobutylicum ATCC 824, E. coli K-12 MG1655, Clostridium beijerinckii NRRL B593, and Thermoanaerobacter brockii HTD4. The strain with the combination of C. acetobutylicum thl (acetyl-coenzyme A [CoA] acetyltransferase), E. coli atoAD (acetoacetyl-CoA transferase), C. acetobutylicum adc (acetoacetate decarboxylase), and C. beijerinckii adh (secondary alcohol dehydrogenase) achieved the highest titer. This strain produced 81.6 mM isopropanol in shake flasks with a yield of 43.5% (mol/mol) in the production phase. To our knowledge, this work is the first to produce isopropanol in E. coli, and the titer exceeded that from the native producers.     

Expression of Solvent-Forming Enzymes and Onset of Solvent Production in Batch Cultures of Clostridium beijerinckii ("Clostridium butylicum")

Clostridium beijerinckii ("Clostridium butylicum") NRRL B592 and NRRL B593 were grown in batch cultures without pH control. The use of more sensitive and accurate procedures for the determination of solvents in cultures led to the recognition of the onset of solvent production about 2 h earlier than the previously assigned point and at a higher culture pH for both strains. Reliable assays for solvent-forming enzyme activities in cell extracts have also been developed. The results showed that activities of solvent-forming enzymes in strain NRRL B592 started to increase about 1 h before the measured onset of solvent production and that the increase in activities of solvent-forming enzymes was not simultaneous. The degree of increase of these enzyme activities for both strains ranged from 2- to 165-fold, with acetoacetate decarboxylase and butanol-isopropanol dehydrogenase showing the largest activity increases. However, the pattern of increase of enzyme activities differed significantly in the two strains of C. beijerinckii. When an increase in solvent-forming enzyme activities was first detected in strain NRRL B592, the culture pH was at 5.7 and the concentrations of total acetic and butyric acids were 5.2 and 3.6 mM, respectively. For strain NRRL B593, the corresponding pH was 5.5. Thus, the culture conditions immediately preceding the expression of solvent-forming enzyme activities differed significantly from those that have been correlated with the production of solvents at later stages of growth.     

Purification and characterization of a primary-secondary alcohol dehydrogenase from two strains of Clostridium beijerinckii.

Two primary alcohols (1-butanol and ethanol) are major fermentation products of several clostridial species. In addition to these two alcohols, the secondary alcohol 2-propanol is produced to a concentration of about 100 mM by some strains of Clostridium beijerinckii. An alcohol dehydrogenase (ADH) has been purified to homogeneity from two strains (NRRL B593 and NESTE 255) of 2-propanol-producing C. beijerinckii. When exposed to air, the purified ADH was stable, whereas the partially purified ADH was inactivated. The ADHs from the two strains had similar structural and kinetic properties. Each had a native M(r) of between 90,000 and 100,000 and a subunit M(r) of between 38,000 and 40,000. The ADHs were NADP(H) dependent, but a low level of NAD(+)-linked activity was detected. They were equally active in reducing aldehydes and 2-ketones, but a much lower oxidizing activity was obtained with primary alcohols than with secondary alcohols. The kcat/Km value for the alcohol-forming reaction appears to be a function of the size of the larger alkyl substituent on the carbonyl group. ADH activities measured in the presence of both acetone and butyraldehyde did not exceed activities measured with either substrate present alone, indicating a common active site for both substrates. There was no similarity in the N-terminal amino acid sequence between that of the ADH and those of fungi and several other bacteria. However, the N-terminal sequence had 67% identity with those of two other anaerobes, Thermoanaerobium brockii and Methanobacterium palustre. Furthermore, conserved glycine and tryptophan residues are present in ADHs of these three anaerobic bacteria and ADHs of mammals and green plants.     

Introducing a single secondary alcohol dehydrogenase into butanol-tolerant Clostridium acetobutylicum Rh8 switches ABE fermentation to high level IBE fermentation

ABSTRACT: BACKGROUND: Previously we have developed a butanol tolerant mutant of Clostridium acetobutylicum, Rh8, from the wild type strain DSM 1731. Strain Rh8 can tolerate up to 19 g/L butanol, with solvent titer improved accordingly, thus exhibiting industrial application potential. To test if strain Rh8 can be used for production of high level mixed alcohols, a single secondary alcohol dehydrogenase from Clostridium beijerinckii NRRL B593 was overexpressed in strain Rh8 under the control of constitutive thl promoter. RESULTS: The heterogenous gene sADH was functionally expressed in C. acetobutylicum Rh8. This simple, one-step engineering approach led to the complete conversion of acetone into isopropanol, achieving a total alcohol titer of 23.88 g/l (7.6 g/l isopropanol, 15 g/l butanol, and 1.28 g/l ethanol) with a yield to glucose of 31.42%. The acid (butyrate and acetate) assimilation rate in isopropanol producing strain Rh8(psADH) was increased. CONCLUSIONS: The improved butanol tolerance and the enhanced solvent biosynthesis machinery in strain Rh8 is beneficial for production of high concentration of mixed alcohols. Strain Rh8 thus can be considered as a good host for further engineering of solvent/alcohol production.     

Genetic systems development in the clostridia

Abstract: This review describes recent developments in the genetic manipulation of the solventogenic clostridia, Clostridium acetobutylicum and C. beijerinckii . It is to be noted that our laboratory stock of C. acetobutylicum ATCC 824, which was obtained from the American Type Culture Collection, has recently been re-identified as C. beijerinckii NCIMB 8052 based on DNA similarity studies using the S1 nuclease method (personal communication, Dr. Jiann-Shin Chen, Virginia Polytechnic Institute and State University). Reference to our laboratory 824 culture has been changed to C. beijerinckii NCIMB 8052 throughout this paper in order to be consistent with this finding. The focus of this review specifically involves the characterization of an M13-like genetic system for the clostridia based on the pCAK1 phagemid, as well as preliminary work on development of a plasmid-based vector based on the indigenous pDM11 plasmid recovered from C. acetobutylicum NCIB 6443. The construction of a C. beijerinckii strain with amplified endoglucanase activity was achieved by inserting the engB gene from C. cellulovorans into C. beijerinckii . The successful expression of a heterologous engB gene from C. cellulovorans in C. beijerinckii NCIMB 8052 has important industrial significance for the eventual utilization of cellulose by this acetone-butanol-ethanol fermentation microorganism.     

Expression of Solvent-Forming Enzymes and Onset of Solvent Production in Batch Cultures of Clostridium beijerinckii (“Clostridium butylicum”)

Clostridium beijerinckii (“Clostridium butylicum”) NRRL B592 and NRRL B593 were grown in batch cultures without pH control. The use of more sensitive and accurate procedures for the determination of solvents in cultures led to the recognition of the onset of solvent production about 2 h earlier than the previously assigned point and at a higher culture pH for both strains. Reliable assays for solvent-forming enzyme activities in cell extracts have also been developed. The results showed that activities of solvent-forming enzymes in strain NRRL B592 started to increase about 1 h before the measured onset of solvent production and that the increase in activities of solvent-forming enzymes was not simultaneous. The degree of increase of these enzyme activities for both strains ranged from 2- to 165-fold, with acetoacetate decarboxylase and butanol-isopropanol dehydrogenase showing the largest activity increases. However, the pattern of increase of enzyme activities differed significantly in the two strains of C. beijerinckii. When an increase in solvent-forming enzyme activities was first detected in strain NRRL B592, the culture pH was at 5.7 and the concentrations of total acetic and butyric acids were 5.2 and 3.6 mM, respectively. For strain NRRL B593, the corresponding pH was 5.5. Thus, the culture conditions immediately preceding the expression of solvent-forming enzyme activities differed significantly from those that have been correlated with the production of solvents at later stages of growth.     

Molecular Cloning, Characterization, and Potential Roles of Cytosolic and Mitochondrial Aldehyde Dehydrogenases in Ethanol Metabolism in Saccharomyces cerevisiae

The full-length DNAs for two Saccharomyces cerevisiae aldehyde dehydrogenase (ALDH) genes were cloned and expressed in Escherichia coli. A 2,744-bp DNA fragment contained an open reading frame encoding cytosolic ALDH1, with 500 amino acids, which was located on chromosome XVI. A 2,661-bp DNA fragment contained an open reading frame encoding mitochondrial ALDH5, with 519 amino acids, of which the N-terminal 23 amino acids were identified as the putative leader sequence. The ALDH5 gene was located on chromosome V. The commercial ALDH (designated ALDH2) was partially sequenced and appears to be a mitochondrial enzyme encoded by a gene located on chromosome XV. The recombinant ALDH1 enzyme was found to be essentially NADP dependent, while the ALDH5 enzyme could utilize either NADP or NAD as a cofactor. The activity of ALDH1 was stimulated two- to fourfold by divalent cations but was unaffected by K⁺ ions. In contrast, the activity of ALDH5 increased in the presence of K⁺ ions: 15-fold with NADP and 40-fold with NAD, respectively. Activity staining of isoelectric focusing gels showed that cytosolic ALDH1 contributed 30 to 70% of the overall activity, depending on the cofactor used, while mitochondrial ALDH2 contributed the rest. Neither ALDH5 nor the other ALDH-like proteins identified from the genomic sequence contributed to the in vitro oxidation of acetaldehyde. To evaluate the physiological roles of these three ALDH isoenzymes, the genes encoding cytosolic ALDH1 and mitochondrial ALDH2 and ALDH5 were disrupted in the genome of strain TWY397 separately or simultaneously. The growth of single-disruption Δald1 and Δald2 strains on ethanol was marginally slower than that of the parent strain. The Δald1 Δald2 double-disruption strain failed to grow on glucose alone, but growth was restored by the addition of acetate, indicating that both ALDHs might catalyze the oxidation of acetaldehyde produced during fermentation. The double-disruption strain grew very slowly on ethanol. The role of mitochondrial ALDH5 in acetaldehyde metabolism has not been defined but appears to be unimportant.     

Expression of plasmid-encoded aad in Clostridium acetobutylicum M5 restores vigorous butanol production.

Mutant M5 of Clostridium acetobutylicum ATCC 824, which produces neither butanol nor acetone and is deficient in butyraldehyde dehydrogenase (BYDH), acetoacetate decarboxylase, and acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase activities, was transformed with plasmid pCAAD, which carries the gene aad (R. V. Nair, G. N. Bennett, and E. T. Papoutsakis, J. Bacteriol, 176:871-885, 1994). In batch fermentation studies, aad expression restored butanol formation (84 mM) in mutant M5 without any acetone formation or any significant increase in ethanol production. The corresponding protein (AAD) appeared as a ca. 96-kDa band in a denaturing protein gel. Expression of AAD in M5 resulted in restoration of BYDH activity and small increases in the activities of acetaldehyde dehydrogenase, butanol dehydrogenase, and ethanol dehydrogenase. These findings suggest that BYDH activity in C. acetobutylicum ATCC 824 resides largely in AAD, and that AAD's primary role is in the formation of butanol rather than of ethanol.     

Molecular analysis and nucleotide sequence of the adh1 gene encoding an NADPH-dependent butanol dehydrogenase in the Gram-positive anaerobe Clostridium acetobutylicum

The nucleotide sequence of a 2081-bp fragment of Clostridium acetobutylicum DNA containing the adh1 gene was determined. The butanol dehydrogenase gene is referred to as the adh1 gene since it was shown to have activity using butanol and ethanol as substrates. The adh1 gene consisted of 1164 bp and encoded an alcohol dehydrogenase (ADH) enzyme of 388 aa residues with an Mr of 43,274. The adh1 gene was separated from an upstream open reading frame by an intergenic region of 354 bp. No promoter consensus sequences were identified in the intergenic upstream region and the adh1 gene did not appear to be expressed off its own promoter in Escherichia coli. Three separate types of ADH have been recognized. The ADH1 from C. acetobutylicum exhibited 39% homology with the Fe-containing ADH2 from Zymomonas mobilis and 37% homology with the ADH4 from Saccharomyces cerevisiae, but showed little or no homology with the other characterised types of ADH.