Efficient production of acetoin in Saccharomyces cerevisiae by disruption of 2,3-butanediol dehydrogenase and expression of NADH oxidase

Farnesyl diphosphate (FPP)-derived isoprenoids represent a diverse group of plant secondary metabolites with great economic potential. To enable their efficient production in the heterologous host Saccharomyces cerevisiae, we refined a metabolic engineering strategy using the CRISPR/Cas9 system with the aim of increasing the availability of FPP for downstream reactions. The strategy included the overexpression of mevalonate pathway (MVA) genes, the redirection of metabolic flux towards desired product formation and the knockout of genes responsible for competitive reactions. Following the optimisation of culture conditions, the availability of the improved FPP biosynthesis for downstream reactions was demonstrated by the expression of a germacrene synthase from dandelion. Subsequently, biosynthesis of significant amounts of germacrene-A was observed in the most productive strain compared to the wild type. Thus, the presented strategy is an excellent tool to increase FPP-derived isoprenoid biosynthesis in yeast.

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Cofactor engineering in Saccharomyces cerevisiae: Expression of a H2O-forming NADH oxidase and impact on redox metabolism

Metabolic Engineering ◽

10.1016/j.ymben.2005.12.003 ◽

2006 ◽

Vol 8 (4) ◽

pp. 303-314 ◽

Cited By ~ 103

Author(s):

Stéphanie Heux ◽

Rémy Cachon ◽

Sylvie Dequin

Keyword(s):

Saccharomyces Cerevisiae ◽

Nadh Oxidase ◽

Cofactor Engineering ◽

Redox Metabolism

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Efficient Production of Ethanol from Empty Palm Fruit Bunch Fibers by Fed-Batch Simultaneous Saccharification and Fermentation Using Saccharomyces cerevisiae

Applied Biochemistry and Biotechnology ◽

10.1007/s12010-013-0314-z ◽

2013 ◽

Vol 170 (8) ◽

pp. 1807-1814 ◽

Cited By ~ 32

Author(s):

Jang Min Park ◽

Baek-Rock Oh ◽

Jeong-Woo Seo ◽

Won-Kyung Hong ◽

Anna Yu ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Simultaneous Saccharification And Fermentation ◽

Efficient Production ◽

Fed Batch ◽

Palm Fruit ◽

Simultaneous Saccharification

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Overexpression of a Water-Forming NADH Oxidase Improves the Metabolism and Stress Tolerance of Saccharomyces cerevisiae in Aerobic Fermentation

Frontiers in Microbiology ◽

10.3389/fmicb.2016.01427 ◽

2016 ◽

Vol 7 ◽

Cited By ~ 2

Author(s):

Xinchi Shi ◽

Yanan Zou ◽

Yong Chen ◽

Cheng Zheng ◽

Hanjie Ying

Keyword(s):

Saccharomyces Cerevisiae ◽

Stress Tolerance ◽

Nadh Oxidase ◽

Aerobic Fermentation

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Role of Saccharomyces cerevisiae Oxidoreductases Bdh1p and Ara1p in the Metabolism of Acetoin and 2,3-Butanediol

Applied and Environmental Microbiology ◽

10.1128/aem.01521-09 ◽

2009 ◽

Vol 76 (3) ◽

pp. 670-679 ◽

Cited By ~ 26

Author(s):

Eva González ◽

M. Rosario Fernández ◽

Didac Marco ◽

Eduard Calam ◽

Lauro Sumoy ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Type Strain ◽

Wild Type Strain ◽

Growth Conditions ◽

Wild Type ◽

Genome Expression ◽

Whole Genome Expression ◽

Histidine Tag ◽

Butanediol Dehydrogenase

ABSTRACT NAD-dependent butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae reversibly transforms acetoin to 2,3-butanediol in a stereospecific manner. Deletion of BDH1 resulted in an accumulation of acetoin and a diminution of 2,3-butanediol in two S. cerevisiae strains under two different growth conditions. The concentrations of (2R,3R)-2,3-butanediol are mostly dependent on Bdh1p activity, while those of (meso)-2,3-butanediol are also influenced by the activity of NADP(H)-dependent oxidoreductases. One of them has been purified and shown to be d-arabinose dehydrogenase (Ara1p), which converts (R/S)-acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol. Deletion of BDH2, a gene adjacent to BDH1, whose encoded protein is 51% identical to Bdh1p, does not significantly alter the levels of acetoin or 2,3-butanediol in comparison to the wild-type strain. Furthermore, we have expressed Bdh2p with a histidine tag and have shown it to be inactive toward 2,3-butanediol. A whole-genome expression analysis with microarrays demonstrates that BDH1 and BDH2 are reciprocally regulated.

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Decreased Xylitol Formation during Xylose Fermentation in Saccharomyces cerevisiae Due to Overexpression of Water-Forming NADH Oxidase

Applied and Environmental Microbiology ◽

10.1128/aem.06635-11 ◽

2011 ◽

Vol 78 (4) ◽

pp. 1081-1086 ◽

Cited By ~ 34

Author(s):

Guo-Chang Zhang ◽

Jing-Jing Liu ◽

Wen-Tao Ding

Keyword(s):

Saccharomyces Cerevisiae ◽

Xylose Fermentation ◽

Nadh Oxidase ◽

Xylose Reductase ◽

Control Strain ◽

Content Type ◽

Ethanol Yields ◽

Glycerol Yield ◽

Set Up ◽

And Control

ABSTRACTThe recombinant xylose-fermentingSaccharomyces cerevisiaestrain harboring xylose reductase (XR) and xylitol dehydrogenase (XDH) fromScheffersomyces stipitisrequires NADPH and NAD+, creates cofactor imbalance, and causes xylitol accumulation during growth ond-xylose. To solve this problem,noxE, encoding a water-forming NADH oxidase fromLactococcus lactisdriven by thePGK1promoter, was introduced into the xylose-utilizing yeast strain KAM-3X. A cofactor microcycle was set up between the utilization of NAD+by XDH and the formation of NAD+by water-forming NADH oxidase. Overexpression ofnoxEsignificantly decreased xylitol formation and increased final ethanol production during xylose fermentation. Under xylose fermentation conditions with an initiald-xylose concentration of 50 g/liter, the xylitol yields for of KAM-3X(pPGK1-noxE) and control strain KAM-3X were 0.058 g/g xylose and 0.191 g/g, respectively, which showed a 69.63% decrease owing tonoxEoverexpression; the ethanol yields were 0.294 g/g for KAM-3X(pPGK1-noxE) and 0.211 g/g for the control strain KAM-3X, which indicated a 39.33% increase due tonoxEoverexpression. At the same time, the glycerol yield also was reduced by 53.85% on account of the decrease in the NADH pool caused by overexpression ofnoxE.

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