Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacteriumsaccharolyticum

Abstract

Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic bacteria that have been engineered to produce ethanol from the cellulose and hemicellulose fractions of biomass, respectively. Although engineered strains of T. saccharolyticum produce ethanol with a yield of 90% of the theoretical maximum, engineered strains of C. thermocellum produce ethanol at lower yields (ϳ50% of the theoretical maximum). In the course of engineering these strains, a number of mutations have been discovered in their adhE genes, which encode both alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. To understand the effects of these mutations, the adhE genes from six strains of C. thermocellum and T. saccharolyticum were cloned and expressed in Escherichia coli, the enzymes produced were purified by affinity chromatography, and enzyme activity was measured. In wild-type strains of both organisms, NADH was the preferred cofactor for both ALDH and ADH activities. In high-ethanol-producing (ethanologen) strains of T. saccharolyticum, both ALDH and ADH activities showed increased NADPH-linked activity. Interestingly, the AdhE protein of the ethanologenic strain of C. thermocellum has acquired high NADPH-linked ADH activity while maintaining NADH-linked ALDH and ADH activities at wild-type levels. When single amino acid mutations in AdhE that caused increased NADPH-linked ADH activity were introduced into C. thermocellum and T. saccharolyticum, ethanol production increased in both organisms. Structural analysis of the wild-type and mutant AdhE proteins was performed to provide explanations for the cofactor specificity change on a molecular level. IMPORTANCEThis work describes the characterization of the AdhE enzyme from different strains of C. thermocellum and T. saccharolyticum. C. thermocellum and T. saccharolyticum are thermophilic anaerobes that have been engineered to make high yields of ethanol and can solubilize components of plant biomass and ferment the sugars to ethanol. In the course of engineering these strains, several mutations arose in the bifunctional ADH/ALDH protein AdhE, changing both enzyme activity and cofactor specificity. We show that changing AdhE cofactor specificity from mostly NADH linked to mostly NADPH linked resulted in higher ethanol production by C. thermocellum and T. saccharolyticum. T hermophilic organisms, Clostridium thermocellum in particular, hold great promise for the production of ethanol from lignocellulosic feedstocks (1, 2). C. thermocellum is a thermophilic, Gram-positive obligate anaerobe that rapidly consumes cellulose. Engineered strains of Thermoanaerobacterium saccharolyticum (3), a thermophilic anaerobe that ferments xylan and other sugars derived from biomass, have been shown to produce ethanol at Ͼ50 g/liter, a near-theoretical yield (4). While comparable concentrations of ethanol are tolerated by selected strains of C. thermocellum (5-7), the maximum reported concentration of ethanol produced by this organism is 23.6 g/liter (8) and th...

Deletion of nfnAB in Thermoanaerobacterium saccharolyticum and Its Effect on Metabolism

“…Unfortunately, genetic manipulation of nfnAB was impossible in ALK2, as ALK2 already has the Kan and Erm resistance markers, which are the only two antibiotic resistance genes available for T. saccharolyticum. Both ALK2 and M1442 contain mutations in adhE that have been shown to change the cofactor specificity of AdhE from primarily NADH linked to NADPH linked (20). In contrast, the wild-type, LL1144, and LL1145 strains appear to use the NADH-linked ethanol production pathway.…”

“…Although mutations in adhE have been shown to produce NADPH-linked ADH activity (20,22,23), another possible source of NADPH-linked ADH activity is the adhA gene, Tsac_2087. It has been shown that in a T. saccharolyticum adhE deletion strain, there were still significant levels of NADPH-linked ADH activity, suggesting that there may be other functional NADPH-linked alcohol dehydrogenases (5).…”

Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA , Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

“…As presented below, there is evidence suggesting multiple enzymes could perform the ADH reaction in T. saccharolyticum, and AdhE may be necessary only for its ALDH function. We have previously reported the biochemical properties of the C. thermocellum and T. saccharolyticum bifunctional AdhE: wild-type (wt) AdhE is mostly NADH linked for ADH activity in both organisms (12). Deleting adhE in C. thermocellum eliminated Ͼ90% of the ADH activity in cell extracts, suggesting that AdhE is the primary enzyme contributing to ADH activity.…”

Ferredoxin:NAD + Oxidoreductase of Thermoanaerobacterium saccharolyticum and Its Role in Ethanol Formation

“…All chemicals were reagent grade and obtained from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA) unless indicated otherwise. CTFUD rich medium at pH 7.0 and pH 6.0 was used for C. thermocellum and T. saccharolyticum, respectively (22,23). The growth temperature was 55°C for both strains.…”

“…Since the alcohol dehydrogenase and acetaldehyde dehydrogenase reactions in C. thermocellum are NADH linked (Fig. 4, NADH based) (22), the NADH-FNOR is more suitable for cofactor balance in C. thermocellum (as opposed to the NADPH-linked NfnAB complex). To test this hypothesis, the tsac_1705 gene was inserted into plasmid pDGO126 (38), and the resulting plasmid was transformed into C. thermocellum.…”