Print Email Facebook Twitter Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism Title Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism Author Marques, W.L. (TU Delft BT/Industrial Microbiology; University of Campinas) Mans, R. (TU Delft BT/Industrial Microbiology) Marella, Eko Roy (Student TU Delft) Cordeiro, Rosa Lorizolla (University of Campinas) van den Broek, M.A. (TU Delft BT/Industrial Microbiology) Daran, J.G. (TU Delft BT/Industrial Microbiology) Pronk, J.T. (TU Delft BT/Industrial Microbiology) Gombert, Andreas K. (University of Campinas) van Maris, A.J.A. (TU Delft BT/Industrial Microbiology) Date 2017 Abstract Many relevant options to improve efficacy and kinetics of sucrose metabolism in Saccharomyces cerevisiae and, thereby, the economics of sucrose-based processes remain to be investigated. An essential first step is to identify all native sucrose-hydrolysing enzymes and sucrose transporters in this yeast, including those that can be activated by suppressor mutations in sucrose-negative strains. A strain in which all known sucrose-transporter genes (MAL11, MAL21, MAL31, MPH2, MPH3) were deleted did not grow on sucrose after 2 months of incubation. In contrast, a strain with deletions in genes encoding sucrose-hydrolysing enzymes (SUC2, MAL12, MAL22, MAL32) still grew on sucrose. Its specific growth rate increased from 0.08 to 0.25 h-1 after sequential batch cultivation. This increase was accompanied by a 3-fold increase of in vitro sucrose-hydrolysis and isomaltase activities, as well as by a 3- to 5-fold upregulation of the isomaltase-encoding genes IMA1 and IMA5. One-step Cas9-mediated deletion of all isomaltase-encoding genes (IMA1-5) completely abolished sucrose hydrolysis. Even after 2 months of incubation, the resulting strain did not grow on sucrose. This sucrose-negative strain can be used as a platform to test metabolic engineering strategies and for fundamental studies into sucrose hydrolysis or transport. Subject DisaccharideIsomaltaseLaboratory evolutionMultiple gene deletionReal-time PCRReverse engineering To reference this document use: http://resolver.tudelft.nl/uuid:da98a321-fc15-4df8-bd83-e7d16ea1420a DOI https://doi.org/10.1093/femsyr/fox006 ISSN 1567-1356 Source FEMS Yeast Research, 17 (1) Part of collection Institutional Repository Document type journal article Rights © 2017 W.L. Marques, R. Mans, Eko Roy Marella, Rosa Lorizolla Cordeiro, M.A. van den Broek, J.G. Daran, J.T. Pronk, Andreas K. Gombert, A.J.A. van Maris Files PDF fox006.pdf 1.15 MB Close viewer /islandora/object/uuid:da98a321-fc15-4df8-bd83-e7d16ea1420a/datastream/OBJ/view