Evolutionary engineering of a glycerol‐3‐phosphate dehydrogenase‐negative, acetate‐reducing Saccharomyces cerevisiae strain enables anaerobic growth at high glucose concentrations. Issue 1 (4th September 2013)

• Record Type: Journal Article
• Authors: Guadalupe‐Medina, Víctor; Metz, Benjamin; Oud, Bart; van, Charlotte M.; Mans, Robert; Pronk, Jack T.; van, Antonius J. A.
• Is Part Of: Microbial biotechnology. Volume 7:Issue 1(2014:Jan.)
• Page Start: 44
• Page End: 53
• Publication Date: 2013-09-04
• Subjects: Microbial biotechnology -- Periodicals; Biotechnology; Microbiology; 660.62
• DOI: 10.1111/1751-7915.12080
• Languages: English
• ISSNs: 1751-7915

Abstract

<abstract abstract-type="main"> <title>Summary</title> <p>Glycerol production by <italic>Saccharomyces cerevisiae</italic>, which is required for redox‐cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic <italic>S. cerevisiae</italic> cultures was eliminated by expressing <italic>Escherichia coli</italic> (acetylating) acetaldehyde dehydrogenase (encoded by <italic>mhpF</italic>) and simultaneously deleting the <italic>GPD1</italic> and <italic>GPD2</italic> genes encoding glycerol‐3‐phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd<sup>–</sup> 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<sup>–</sup><italic>mhpF</italic>‐expressing <italic>S. cerevisiae</italic> 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<sup>−1</sup>. The evolved strain produced glycerol at low concentrations (0.64 ± 0.33 g l<sup>−1</sup>). However, these glycerol concentrations were below 10% of those observed with a Gpd<sup>+</sup> 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 <italic>mhpF</italic> gene for anaerobic growth.</p> </abstract>