Determining the roles of the three alcohol dehydrogenases (AdhA, AdhB and AdhE) in Thermoanaerobacter ethanolicus during ethanol formation
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Thermoanaerobacter ethanolicus is a promising candidate for biofuel production due to the broad range of substrates it can utilize and its high ethanol yield compared to other thermophilic bacteria, such as Clostridium thermocellum. Three alcohol dehydrogenases, AdhA, AdhB and AdhE, play key roles in ethanol formation. To study their physiological roles during ethanol formation, we deleted them separately and in combination. Previously, it has been thought that both AdhB and AdhE were bifunctional alcohol dehydrogenases. Here we show that AdhE has primarily acetyl-CoA reduction activity (ALDH) and almost no acetaldehyde reduction (ADH) activity, whereas AdhB has no ALDH activity and but high ADH activity. We found that AdhA and AdhB have similar patterns of activity. Interestingly, although deletion of both adhA and adhB reduced ethanol production, a single deletion of either one actually increased ethanol yields by 60–70%.
KeywordsBioethanol Gene deletion Thermophilic bacteria Bifunctional alcohol dehydrogenase
We thank Marybeth I. Maloney for preparing genomic DNA for resequencing and Dr. Johannes P. van Dijken for providing valuable suggestions and comments for experiments.
The BioEnergy Science Center is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science.
The genomic resequencing work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.
Notice: This manuscript has been authored by Dartmouth College under contract No. DE-AC05-00OR22725 with U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable world-wide license to publish or reproduce the published form of this manuscript or allow others to do so, for United States Government purposes. (End of Notice).
- 1.Argyros DA, Tripathi SA, Barrett TF, Rogers SR, Feinberg LF, Olson DG, Foden JM, Miller BB, Lynd LR, Hogsett DA, Caiazza NC (2011) High ethanol titers from cellulose by using metabolically engineered thermophilic, anaerobic microbes. Appl Environ Microbiol 77:8288–8294. doi: 10.1128/AEM.00646-11 CrossRefPubMedPubMedCentralGoogle Scholar
- 3.Biosciences BD (2006) BD Bionutrients ™ technical manual—advanced bioprocessing. Franklin Lakes, NJGoogle Scholar
- 7.Brown SD, Guss AM, Karpinets TV, Parks JM, Smolin N, Yang S, Land ML, Klingeman DM, Bhandiwad A, Rodriguez M, Raman B, Shao X, Mielenz JR, Smith JC, Keller M, Lynd LR (2011) Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum. Proc Natl Acad Sci USA 108:13752–13757. doi: 10.1073/pnas.1102444108 CrossRefPubMedPubMedCentralGoogle Scholar
- 10.Burdette D, Zeikus JG (1994) Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 degrees Adh) as a bifunctional alcohol dehydrogenase–acetyl-CoA reductive. Biochem J 302:163–170CrossRefPubMedPubMedCentralGoogle Scholar
- 12.Carere CR, Rydzak T, Verbeke TJ, Cicek N, Levin DB, Sparling R (2012) Linking genome content to biofuel production yields: a meta-analysis of major catabolic pathways among select H2 and ethanol-producing bacteria. BMC Microbiol 12:295. doi: 10.1186/1471-2180-12-295 CrossRefPubMedPubMedCentralGoogle Scholar
- 17.Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, Potter SC, Punta M, Qureshi M, Sangrador-Vegas A, Salazar GA, Tate J, Bateman A (2015) The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 44:D279–D285. doi: 10.1093/nar/gkv1344 CrossRefPubMedPubMedCentralGoogle Scholar
- 20.Hemme CL, Fields MW, He Q, Deng Y, Lin L, Tu Q, Mouttaki H, Zhou A, Feng X, Zuo Z, Ramsay BD, He Z, Wu L, Van Nostrand J, Xu J, Tang YJ, Wiegel J, Phelps TJ, Zhou J (2011) Correlation of genomic and physiological traits of Thermoanaerobacter species with biofuel yields. Appl Environ Microbiol 77:7998–8008. doi: 10.1128/AEM.05677-11 CrossRefPubMedPubMedCentralGoogle Scholar
- 21.Herring CD, Kenealy WR, Shaw AJ, Raman B, Tschaplinski TJ, Brown SD, Davison BH, Covalla SF, Sillers WR, Xu H, Tsakraklides V, Hogsett DA (2012) Final report on development of Thermoanaerobacterium saccharolyticum for the conversion of lignocellulose to ethanol. doi: 10.2172/1033560Google Scholar
- 27.Lo J, Zheng T, Hon S, Olson DG, Lynd LR (2015) The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is necessary for ethanol production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum. J Bacteriol 197:1386–1393. doi: 10.1128/JB.02450-14 CrossRefPubMedPubMedCentralGoogle Scholar
- 36.Shaw AJ, Hogsett DA, Lynd LR (2009) Identification of the [FeFe]-hydrogenase responsible for hydrogen generation in Thermoanaerobacterium saccharolyticum and demonstration of increased ethanol yield via hydrogenase knockout. J Bacteriol 191:6457–6464. doi: 10.1128/JB.00497-09 CrossRefPubMedPubMedCentralGoogle Scholar
- 42.van der Veen D, Lo J, Brown SD, Johnson CM, Tschaplinski TJ, Martin M, Engle NL, van den Berg RA, Argyros AD, Caiazza NC, Guss AM, Lynd LR (2013) Characterization of Clostridium thermocellum strains with disrupted fermentation end-product pathways. J Ind Microbiol Biotechnol 40:725–734. doi: 10.1007/s10295-013-1275-5 CrossRefPubMedGoogle Scholar
- 43.Wang Q, Wang Q, Tong W, Bai X, Chen Z, Zhao J, Zhang J, Liu S (2012) Regulation of enzyme activity of alcohol dehydrogenase through its interactions with pyruvate-ferredoxin oxidoreductase in Thermoanaerobacter tengcongensis. Biochem Biophys Res Commun 417:1018–1023. doi: 10.1016/j.bbrc.2011.12.083 CrossRefPubMedGoogle Scholar
- 48.Zheng T, Olson DG, Murphy SJ, Shao X, Tian L, Lynd LR (2016) Both adhE and a separate NADPH-dependent alcohol dehydrogenase (adhA) are necessary for high ethanol production in Thermoanaerobacterium saccharolyticum. J Bacteriol: JB00542. doi: 10.1128/JB.00542-16Google Scholar
- 49.Zheng T, Olson DG, Tian L, Bomble YJ, Himmel ME, Lo J, Hon S, Shaw JA, van Dijken JP, Lynd LR (2015) Cofactor specificity of the bifunctional alcohol and aldehyde dehydrogenase (AdhE) in wild-type and mutants of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum. J Bacteriol 197:2610–2619. doi: 10.1128/JB.00232-15 CrossRefPubMedPubMedCentralGoogle Scholar
- 51.Zhou J, Olson DG, Lanahan AA, Tian L, Murphy SJ-L, Lo J, Lynd LR (2015) Physiological roles of pyruvate ferredoxin oxidoreductase and pyruvate formate-lyase in Thermoanaerobacterium saccharolyticum JW/SL-YS485. Biotechnol Biofuels 8:138. doi: 10.1186/s13068-015-0304-1 CrossRefPubMedPubMedCentralGoogle Scholar