BioChem - DOS

Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae

Metabolic engineering

Marques, Wesley Leoricy; Mans, Robert; Henderson, Ryan K.; Marella, Eko Roy; Horst, Jolanda ter; Hulster, Erik de; Poolman, Bert; Daran, Jean-Marc; Pronk, Jack T.; Gombert, Andreas K.; van Maris, Antonius J.A.

<P><B>Abstract</B></P> <P>Anaerobic industrial fermentation processes do not require aeration and intensive mixing and the accompanying cost savings are beneficial for production of chemicals and fuels. However, the free-energy conservation of fermentative pathways is often insufficient for the production and export of the desired compounds and/or for cellular growth and maintenance. To increase free-energy conservation during fermentation of the industrially relevant disaccharide sucrose by <I>Saccharomyces cerevisiae</I>, we first replaced the native yeast &alpha;-glucosidases by an intracellular sucrose phosphorylase from <I>Leuconostoc mesenteroides</I> (<I>Lm</I>SPase). Subsequently, we replaced the native proton-coupled sucrose uptake system by a putative sucrose facilitator from <I>Phaseolus vulgaris</I> (PvSUF1). The resulting strains grew anaerobically on sucrose at specific growth rates of 0.09 &plusmn; 0.02h<SUP>&minus;1</SUP> (<I>LmSPase</I>) and 0.06 &plusmn; 0.01h<SUP>&minus;1</SUP> (<I>PvSUF1</I>, <I>LmSPase</I>). Overexpression of the yeast <I>PGM2</I> gene, which encodes phosphoglucomutase, increased anaerobic growth rates on sucrose of these strains to 0.23 &plusmn; 0.01h<SUP>&minus;1</SUP> and 0.08 &plusmn; 0.00h<SUP>&minus;1</SUP>, respectively. Determination of the biomass yield in anaerobic sucrose-limited chemostat cultures was used to assess the free-energy conservation of the engineered strains. Replacement of intracellular hydrolase with a phosphorylase increased the biomass yield on sucrose by 31%. Additional replacement of the native proton-coupled sucrose uptake system by PvSUF1 increased the anaerobic biomass yield by a further 8%, resulting in an overall increase of 41%. By experimentally demonstrating an energetic benefit of the combined engineering of disaccharide uptake and cleavage, this study represents a first step towards anaerobic production of compounds whose metabolic pathways currently do not conserve sufficient free-energy.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Functional replacement of native <I>S. cerevisiae</I> sucrose hydrolysis and uptake. </LI> <LI> Replacement of sucrose hydrolysis by phosphorolysis increased biomass yield by 31%. </LI> <LI> Replacement of sucrose/H<SUP>+</SUP>-symport (Mal11) by PvSUF1 further increased yield by 8%. </LI> <LI> Overexpression of <I>PGM2</I> increased the growth rate of <I>Lm</I>SPase expressing strains. </LI> </UL> </P>

2018

Elsevier

1096-7176

1096-7184

45

pp.121-133

10.1016/j.ymben.2017.11.012

http://click.ndsl.kr/servlet/OpenAPIDetailView?keyValue=05787966&target=NART&cn=NART80064243

https://pure.rug.nl/ws/files/51456948/1_s2.0_S109671761730191X_main.pdf

Free-energy conservation; ATP; Facilitated diffusion; Phosphoglucomutase; Chemostat; Yeast physiology