"uuid","repository link","title","author","contributor","publication year","abstract","subject topic","language","publication type","publisher","isbn","issn","patent","patent status","bibliographic note","access restriction","embargo date","faculty","department","research group","programme","project","coordinates" "uuid:93a40aff-930b-4dff-9769-3410ba0e5519","http://resolver.tudelft.nl/uuid:93a40aff-930b-4dff-9769-3410ba0e5519","Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae","Marques, W.L. (TU Delft BT/Industriele Microbiologie; University of Campinas; Universidade de São Paulo); Mans, R. (TU Delft BT/Industriele Microbiologie); Henderson, Ryan K. (Rijksuniversiteit Groningen); Marella, Eko Roy; ter Horst, J. (TU Delft BT/Industriele Microbiologie); de Hulster, A.F. (TU Delft BT/Industriele Microbiologie); Poolman, Bert (Rijksuniversiteit Groningen); Daran, J.G. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie); Gombert, Andreas K. (University of Campinas); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie; AlbaNova University Center)","","2018","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.","ATP; Chemostat; Facilitated diffusion; Free-energy conservation; Phosphoglucomutase; Yeast physiology","en","journal article","","","","","","Accepted Author Manuscript","","2018-12-18","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""