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

• 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 Saccharomyces cerevisiae, we first replaced the native yeast α-glucosidases by an intracellular sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase). Subsequently, we replaced the native proton-coupled sucrose uptake system by a putative sucrose facilitator from Phaseolus vulgaris (PvSUF1). The resulting strains grew anaerobically on sucrose at specific growth rates of 0.09 ± 0.02 h−1 (LmSPase) and 0.06 ± 0.01 h−1 (PvSUF1, LmSPase). Overexpression of the yeast PGM2 gene, which encodes phosphoglucomutase, increased anaerobic growth rates on sucrose of these strains to 0.23 ± 0.01 h−1 and 0.08 ± 0.00 h−1, 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.

Ämnesord

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik -- Bioenergi (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology -- Bioenergy (hsv//eng)

Nyckelord

ATP

Chemostat

Facilitated diffusion

Free-energy conservation

Phosphoglucomutase

Yeast physiology

Adenosinetriphosphate

Biomass

Chemostats

Ecology

Energy conservation

Enzyme activity

Fermentation

Free energy

Industrial chemicals

Phosphorylation

Yeast

Facilitated diffusions

Industrial fermentation

Leuconostoc mesenteroides

Phaseolus vulgaris

Specific growth rate

Sucrose phosphorylase

Yeast physiologies

Sugar (sucrose)

adenosine triphosphate

disaccharide

glucose 6 phosphate

hydrolase

phosphorylase

sucrose

alkalinization

anaerobic growth

Arabidopsis thaliana

Article

carbon source

cell suspension

cellular distribution

chemical reaction

controlled study

fungus growth

genetic background

metabolic engineering

microscopy

molecular biology

nonhuman

pea

priority journal

protein content

proton motive force

rice

Saccharomyces cerevisiae

sucrose metabolism

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)