The aldehyde/alcohol dehydrogenase (AdhE) in relation to the ethanol formation in Thermoanaerobacter ethanolicus JW200

Peng, Hui; Wu, Guogan; Shao, Weilan

doi:10.1016/j.anaerobe.2007.09.004

Cited by 43 publications

(35 citation statements)

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“…Both strains also encode a bifunctional secondary alcohol dehydrogenase/aldehyde dehydrogenase (adhE) ( Fig. 2; Table 1) implicated in ethanol production in Thermoanaerobacter (32) and in butanol production in Clostridium acetobutylicum (11). This complement of ADH enzymes is novel compared to that of most ethanol-producing bacteria, which typically employ a primary ADH (adhA) as the terminal step in ethanol production (4).…”

Section: Resultsmentioning

confidence: 99%

“…The study of Thermoanaerobacter species has long been driven by interest in the ability to efficiently produce ethanol at high yields from both glucose and xylose (21,32,50) and in the potential applications in CBP, a concept offering the most promise in addressing central challenges to cellulosic bioethanol production (23). Several features in particular make Thermoanaerobacter species attractive for integration into CBP schemes, including (i) the ability to efficiently ferment both hexose and pentose sugars (particularly xylose) (21) to ethanol in their natural state, in contrast to non-pentose-fermenting strains such as Zymomonas (38); (ii) relatively high ethanol yields generated from sugar fermentation (14); (iii) simplification of nutrient requirements due to the capability for de novo cofactor (i.e., vitamin B 12 ) biosynthesis; (iv) a high growth temperature which is better suited for industrial processing of ethanol and minimization of microbial contamination than those for mesophilic strains such as engineered E. coli; (v) a high substrate affinity coupled with nearly complete substrate utilization; (vi) ease of growth in microbial consortia; and (vii) a unique terminal ethanol production pathway involving novel lineage-specific alcohol dehydrogenase enzymes which may positively affect fermentation balance and ethanol yields (3a, 4, 32).…”

Section: Discussionmentioning

confidence: 99%

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Correlation of Genomic and Physiological Traits of Thermoanaerobacter Species with Biofuel Yields

Hemme

Fields

et al. 2011

Appl Environ Microbiol

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Thermophilic anaerobic noncellulolytic Thermoanaerobacter species are of great biotechnological importance in cellulosic ethanol production due to their ability to produce high ethanol yields by simultaneous fermentation of hexose and pentose. Understanding the genome structure of these species is critical to improving and implementing these bacteria for possible biotechnological use in consolidated bioprocessing schemes (CBP) for cellulosic ethanol production. Here we describe a comparative genome analysis of two ethanologenic bacteria, Thermoanaerobacter sp. X514 and Thermoanaerobacter pseudethanolicus 39E. Compared to 39E, X514 has several unique key characteristics important to cellulosic biotechnology, including additional alcohol dehydrogenases and xylose transporters, modifications to pentose metabolism, and a complete vitamin B 12 biosynthesis pathway. Experimental results from growth, metabolic flux, and microarray gene expression analyses support genome sequencing-based predictions which help to explain the distinct differences in ethanol production between these strains. The availability of whole-genome sequence and comparative genomic analyses will aid in engineering and optimizing Thermoanaerobacter strains for viable CBP strategies.Global energy demands will increase significantly in coming decades (16), and renewable energy sources such as biofuels have been proposed to help reduce dependence upon fossil energy (8,23,27,49). Current efforts focus on biofuel production from renewable lignocellulosic feedstock (e.g., switchgrass), which constitutes ϳ50% of the world's biomass (2,8,23,27,49). Although intensive research and development have been performed on the effective utilization of lignocellulose, problems associated with practical use of this material have not been resolved fully (13). When enzymatic hydrolysis is adopted for cellulosic ethanol production, different levels of process integration can be visualized: (i) separate (or sequential) hydrolysis and fermentation (SHF), where the enzymes (cellulases) are used separately from fermentation tanks; (ii) simultaneous saccharification and fermentation (SSF), which consolidates enzymatic hydrolysis with fermentation of hexose or pentose; (iii) simultaneous saccharification and cofermentation (SSCF), which further combines the fermentation of hexose and pentose together; and (iv) consolidated bioprocessing (CBP), where all required enzymes and ethanol are produced in a single reactor (1,13,24,29,46,51). While these production schemes represent increasing levels of simplification through process consolidation, consolidation of multiple steps often results in a loss of process efficiency. Thus, improving the efficiency of individual steps, such as cellulose hydrolysis and ethanol fermentation, remains an important task for the development of economically feasible cellulosic bioethanol.Recent efforts have focused on metabolic engineering and (more recently) synthetic biology to produce strains or consortia capable of producing biofuels. Efforts to ...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Correlation of Genomic and Physiological Traits of Thermoanaerobacter Species with Biofuel Yields

Hemme

Fields

et al. 2011

Appl Environ Microbiol

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“…Previously, we demonstrated that adhE is an important gene for ethanol production in both C. thermocellum and T. saccharolyticum: deletion of adhE reduced the ethanol yield by Ͼ95% in both organisms (6). This was not unexpected, since AdhE proteins are essential for anaerobic ethanol production in many organisms (7)(8)(9)(10)(11). However, because AdhE is a bifunctional enzyme with two functional domains, the loss of ethanol formation from the adhE deletion is not evidence that both domains are essential for ethanol production.…”

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confidence: 93%

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

et al. 2017

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Thermoanaerobacterium saccharolyticum has been engineered to produce ethanol at about 90% of the theoretical maximum yield (2 ethanol molecules per glucose equivalent) and a titer of 70 g/liter. Its ethanol-producing ability has drawn attention to its metabolic pathways, which could potentially be transferred to other organisms of interest. Here, we report that the iron-containing AdhA is important for ethanol production in the high-ethanol strain of T. saccharolyticum (LL1049). A single-gene deletion of adhA in LL1049 reduced ethanol production by ϳ50%, whereas multiple gene deletions of all annotated alcohol dehydrogenase genes except adhA and adhE did not affect ethanol production. Deletion of adhA in wild-type T. saccharolyticum reduced NADPH-linked alcohol dehydrogenase (ADH) activity (acetaldehyde-reducing direction) by 93%.IMPORTANCE In this study, we set out to identify the alcohol dehydrogenases necessary for high ethanol production in T. saccharolyticum. Based on previous work, we had assumed that adhE was the primary alcohol dehydrogenase gene. Here, we show that both adhA and adhE are needed for high ethanol yield in the engineered strain LL1049. This is the first report showing adhA is important for ethanol production in a native adhA host, which has important implications for achieving higher ethanol yields in other microorganisms.

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“…1) (12). AdhE is present in a variety of mesophilic and thermophilic anaerobic bacteria capable of producing ethanol as a fermentation product (13)(14)(15)(16). AdhE has also been found in parasitic eukaryotes (17), anaerobic fungi (18), and algae (19).…”

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confidence: 99%

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

et al. 2015

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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...

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The aldehyde/alcohol dehydrogenase (AdhE) in relation to the ethanol formation in Thermoanaerobacter ethanolicus JW200

Cited by 43 publications

References 10 publications

Correlation of Genomic and Physiological Traits of Thermoanaerobacter Species with Biofuel Yields

Correlation of Genomic and Physiological Traits of Thermoanaerobacter Species with Biofuel Yields

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

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

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