Global Gene Expression Patterns in
            <i>Clostridium thermocellum</i>
            as Determined by Microarray Analysis of Chemostat Cultures on Cellulose or Cellobiose

Riederer, Allison; Takasuka, Taichi E.; Makino, Shin-ichi; Stevenson, David; Bukhman, Yury V.; Elsen, Nathaniel L.; Fox, Brian G.

doi:10.1128/aem.02008-10

Cited by 79 publications

(103 citation statements)

References 42 publications

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“…tengcongensis makes ethanol without an annotated adhE (14,15). Transcriptomic and proteomic studies in T. saccharolyticum and C. thermocellum have shown adhE to be highly expressed, although other putative adh genes were detected as well (16)(17)(18)(19). AdhE also has been shown to play a role in ethanol tolerance (20).…”

Section: Importancementioning

confidence: 99%

The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene, adhE , Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

Zheng

Hon

et al. 2015

J Bacteriol

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Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the adhE gene in C. thermocellum and T. saccharolyticum. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In both adhE deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. In T. saccharolyticum, the adhE deletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in the T. saccharolyticum parent strain but did not increase activity in the adhE deletion strain, suggesting that ALDH activity was inhibited. In C. thermocellum, the adhE deletion strain lost >90% of ALDH and ADH activity in cell extracts. The C. thermocellum adhE deletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase. IMPORTANCEThermoanaerobacterium saccharolyticum and Clostridium thermocellum are bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol dehydrogenases which could be responsible for alcohol formation. This study explores the inactivation of adhE, a gene encoding a bifunctional alcohol and aldehyde dehydrogenase. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In strains without adhE, we note changes in biochemical activity, product formation, and growth.

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Section: Importancementioning

confidence: 99%

The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene, adhE , Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

Zheng

Hon

et al. 2015

J Bacteriol

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“…2), and indeed no ethanol production is detected from this strain (18). The phylogenetically related, thermophilic Firmicute C. thermocellum, however, encodes an NADH-dependent AdhE (Cthe0423), which is one of the most highly expressed genes (24) and abundant proteins (25) in C. thermocellum and is the key enzyme in ethanol production in this organism. Based on its known thermostability, coenzyme specificity (NADH-dependent), similarity in codon use, and favorable catalytic stoichiometry (26), this adhE was a promising candidate to generate an ethanol production pathway in C. bescii.…”

Section: Resultsmentioning

confidence: 99%

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

Chung

Cha

Guss

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

192

171

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Ethanol is the most widely used renewable transportation biofuel in the United States, with the production of 13.3 billion gallons in 2012 [John UM (2013) Contribution of the Ethanol Industry to the Economy of the United States]. Despite considerable effort to produce fuels from lignocellulosic biomass, chemical pretreatment and the addition of saccharolytic enzymes before microbial bioconversion remain economic barriers to industrial deployment [Lynd LR, et al. (2008) Nat Biotechnol 26(2):169-172]. We began with the thermophilic, anaerobic, cellulolytic bacterium Caldicellulosiruptor bescii, which efficiently uses unpretreated biomass, and engineered it to produce ethanol. Here we report the direct conversion of switchgrass, a nonfood, renewable feedstock, to ethanol without conventional pretreatment of the biomass. This process was accomplished by deletion of lactate dehydrogenase and heterologous expression of a Clostridium thermocellum bifunctional acetaldehyde/alcohol dehydrogenase. Whereas wild-type C. bescii lacks the ability to make ethanol, 70% of the fermentation products in the engineered strain were ethanol [12.8 mM ethanol directly from 2% (wt/vol) switchgrass, a real-world substrate] with decreased production of acetate by 38% compared with wild-type. Direct conversion of biomass to ethanol represents a new paradigm for consolidated bioprocessing, offering the potential for carbon neutral, cost-effective, sustainable fuel production. metabolic engineering | bioenergy and thermophiles

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“…S6). A recent study has shown that Cthe_0423 ADH transcripts are among the most abundant C. thermocellum transcripts (24). Although there was a decrease in total ADH activity in adhE*(EA), it was still sufficient for ethanol production.…”

Section: Nonrandom Distribution Of Mutations Across the Genome And Theirmentioning

confidence: 99%

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

Brown

Guss

Karpinets

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

160

145

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Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene ( adhE ), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

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Global Gene Expression Patterns in Clostridium thermocellum as Determined by Microarray Analysis of Chemostat Cultures on Cellulose or Cellobiose

Cited by 79 publications

References 42 publications

The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene, adhE , Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene, adhE , Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

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