Affiliation:
1. Mascoma LLC, Lebanon, New Hampshire, USA
Abstract
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.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
35 articles.
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