Print Email Facebook Twitter Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae Title Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae Author 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) Department BT/Biotechnologie Date 2018 Abstract 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. Subject ATPChemostatFacilitated diffusionFree-energy conservationPhosphoglucomutaseYeast physiology To reference this document use: http://resolver.tudelft.nl/uuid:93a40aff-930b-4dff-9769-3410ba0e5519 DOI https://doi.org/10.1016/j.ymben.2017.11.012 Embargo date 2018-12-18 ISSN 1096-7176 Source Metabolic Engineering, 45, 121-133 Bibliographical note Accepted Author Manuscript Part of collection Institutional Repository Document type journal article Rights © 2018 W.L. Marques, R. Mans, Ryan K. Henderson, Eko Roy Marella, J. ter Horst, A.F. de Hulster, Bert Poolman, J.G. Daran, J.T. Pronk, Andreas K. Gombert, A.J.A. van Maris Files PDF 20170922_Sucrose_energeti ... script.pdf 1.38 MB Close viewer /islandora/object/uuid:93a40aff-930b-4dff-9769-3410ba0e5519/datastream/OBJ/view