title: Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.
abstract: Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level.
authors.affiliation: Mascoma LLC, Lebanon, New Hampshire, USA.
authors.forename: Brooks M
authors.initials: BM
authors.lastname: Henningsen
authors.affiliation: Mascoma LLC, Lebanon, New Hampshire, USA.
authors.forename: Shuen
authors.initials: S
authors.lastname: Hon
authors.affiliation: Mascoma LLC, Lebanon, New Hampshire, USA.
authors.forename: Sean F
authors.initials: SF
authors.lastname: Covalla
authors.affiliation: Mascoma LLC, Lebanon, New Hampshire, USA.
authors.forename: Carolina
authors.initials: C
authors.lastname: Sonu
authors.affiliation: Mascoma LLC, Lebanon, New Hampshire, USA.
authors.forename: D Aaron
authors.initials: DA
authors.lastname: Argyros
authors.affiliation: Mascoma LLC, Lebanon, New Hampshire, USA.
authors.forename: Trisha F
authors.initials: TF
authors.lastname: Barrett
authors.affiliation: Mascoma LLC, Lebanon, New Hampshire, USA.
authors.forename: Erin
authors.initials: E
authors.lastname: Wiswall
authors.affiliation: Mascoma LLC, Lebanon, New Hampshire, USA.
authors.forename: Allan C
authors.initials: AC
authors.lastname: Froehlich
authors.affiliation: Mascoma LLC, Lebanon, New Hampshire, USA rzelle@lallemand.com.
authors.forename: Rintze M
authors.identifier: http://orcid.org/0000-0003-1779-8883
authors.initials: RM
authors.lastname: Zelle
mesh_terms: D000085:Acetates / Q000378:metabolism*; D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000693:Anaerobiosis; D056804:Biofuels / Q000032:analysis*; D003067:Coenzymes / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism*; D005285:Fermentation; D005818:Genetic Engineering; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
chemical_list: D000085:Acetates; D056804:Biofuels; D003067:Coenzymes; D009243:NAD; D000431:Ethanol; D009249:NADP; D000426:Alcohol Dehydrogenase