Improving ethanol yield in acetate-reducing Saccharomyces cerevisiae by cofactor engineering of 6-phosphogluconate dehydrogenase and deletion of ALD6

Improving ethanol yield in acetate-reducing Saccharomyces cerevisiae by cofactor engineering of 6-phosphogluconate dehydrogenase and deletion of ALD6

Papapetridis, I. (TU Delft BT/Industriele Microbiologie)
van Dijk, M. (TU Delft BT/Industriele Microbiologie)
Dobbe, Arthur P A
Metz, B. (TU Delft BT/Industriele Microbiologie)
Pronk, J.T. (TU Delft BT/Industriele Microbiologie)
van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie)

2016-04-26

Background: Acetic acid, an inhibitor of sugar fermentation by yeast, is invariably present in lignocellulosic hydrolysates which are used or considered as feedstocks for yeast-based bioethanol production. Saccharomyces cerevisiae strains have been constructed, in which anaerobic reduction of acetic acid to ethanol replaces glycerol formation as a mechanism for reoxidizing NADH formed in biosynthesis. An increase in the amount of acetate that can be reduced to ethanol should further decrease acetic acid concentrations and enable higher ethanol yields in industrial processes based on lignocellulosic feedstocks. The stoichiometric requirement of acetate reduction for NADH implies that increased generation of NADH in cytosolic biosynthetic reactions should enhance acetate consumption. Results: Replacement of the native NADP⁺-dependent 6-phosphogluconate dehydrogenase in S. cerevisiae by a prokaryotic NAD⁺-dependent enzyme resulted in increased cytosolic NADH formation, as demonstrated by a ca. 15 % increase in the glycerol yield on glucose in anaerobic cultures. Additional deletion of ALD6, which encodes an NADP⁺-dependent acetaldehyde dehydrogenase, led to a 39 % increase in the glycerol yield compared to a non-engineered strain. Subsequent replacement of glycerol formation by an acetate reduction pathway resulted in a 44 % increase of acetate consumption per amount of biomass formed, as compared to an engineered, acetate-reducing strain that expressed the native 6-phosphogluconate dehydrogenase and ALD6. Compared to a non-acetate reducing reference strain under the same conditions, this resulted in a ca. 13 % increase in the ethanol yield on glucose. Conclusions: The combination of NAD⁺-dependent 6-phosphogluconate dehydrogenase expression and deletion of ALD6 resulted in a marked increase in the amount of acetate that was consumed in these proof-of-principle experiments, and this concept is ready for further testing in industrial strains as well as in hydrolysates. Altering the cofactor specificity of the oxidative branch of the pentose-phosphate pathway in S. cerevisiae can also be used to increase glycerol production in wine fermentation and to improve NADH generation and/or generation of precursors derived from the pentose-phosphate pathway in other industrial applications of this yeast.

6-phosphogluconate dehydrogenase
Acetic acid
NADH
NADPH
Redox metabolism
Yeast

http://resolver.tudelft.nl/uuid:e59389dc-9b03-4e98-875a-79e290907c41

https://doi.org/10.1186/s12934-016-0465-z

1475-2859

Microbial Cell Factories, 15

Institutional Repository

journal article

© 2016 I. Papapetridis, M. van Dijk, Arthur P A Dobbe, B. Metz, J.T. Pronk, A.J.A. van Maris