Evolutionary engineering of a glycerol‐3‐phosphate dehydrogenase‐negative, acetate‐reducing Saccharomyces cerevisiae strain enables anaerobic growth at high glucose concentrations

Abstract

Glycerol production by Saccharomyces cerevisiae, which is required for redox-cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic S. cerevisiae cultures was eliminated by expressing Escherichia coli (acetylating) acetaldehyde dehydrogenase (encoded by mhpF) and simultaneously deleting the GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd– strains are, however, sensitive to high sugar concentrations, which complicates industrial implementation of this metabolic engineering concept. In this study, laboratory evolution was used to improve osmotolerance of a Gpd– mhpF-expressing S. cerevisiae strain. Serial batch cultivation at increasing osmotic pressure enabled isolation of an evolved strain that grew anaerobically at 1 M glucose, at a specific growth rate of 0.12 h−1. The evolved strain produced glycerol at low concentrations (0.64 ± 0.33 g l−1). However, these glycerol concentrations were below 10% of those observed with a Gpd+ reference strain. Consequently, the ethanol yield on sugar increased from 79% of the theoretical maximum in the reference strain to 92% for the evolved strains. Genetic analysis indicated that osmotolerance under aerobic conditions required a single dominant chromosomal mutation, and one further mutation in the plasmid-borne mhpF gene for anaerobic growth.

Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

“…3), while the ethanol titer increased by 0. A similar increase in glycerol production was observed by Guadalupe Medina et al after evolution of a Gpd Ϫ ADA strain, which was speculated to be due to increased glycerolipid degradation (23,39).…”

“…The acetate consumption strains described in this study have glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2 deleted. While a convenient genetic modification to avoid competition for the anaerobic biosynthetic NADH surplus, Gpd Ϫ strains are known to be sensitive to osmotic stress and to be less robust (23,46). However, a reduction in glycerol formation is still very much desired since it increases the ethanol yield.…”

“…Guadalupe Medina et al reported consumption of 0.022 and 0.012 g acetate (g glucose) Ϫ1 for a nonevolved and an evolved Gpd Ϫ ADA strain, respectively (18,23). Based on the metabolite profile of the parental wild-type strain, the authors calculated a surplus of 7.8 mmol NADH per g CDW produced, which was reoxidized via the consumption of 3.9 mmol (0.23 g) acetate per g CDW.…”

The glycerol and ethanol production kinetics in low‐temperature wine fermentation using Saccharomyces cerevisiae yeast strains

“…The NAD + ‐dependent glycerol‐3‐phosphate dehydrogenase is a key enzyme for glycerol formation in S. cerevisiae (Guadalupe‐Medina et al ., ). The activity of glycerol‐3‐phosphate dehydrogenase (GPD, EC 1.1.1.8) was determined as described (Guadalupe‐Medina et al ., ) with some modifications. The reaction solution consisted of 20 m m Hepes, pH 7.1, 20 m m KCl, 1 m m EDTA, 1 m m DTT, 5 m m dihydroxyacetonephosphate (DHAP) and 0.1 m m NADH.…”

Increased production of L-serine in Escherichia coli through Adaptive Laboratory Evolution

“…ALE is increasingly being used to enhance tolerance of cells towards inhibiting environmental conditions (Arense et al, 2010;Wang and Church, 2011), metabolites (Kwon et al, 2011) and oxygen limited conditions (Guadalupe-Medina et al, 2014). ALE has also been successfully applied for latent pathway activation (Portnoy et al, 2011) and for enhanced production of desired compound (Reyes et al, 2014).…”