title: Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism.
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.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands.|School of Food Engineering, University of Campinas, Campinas, SP 13083-862, Brazil.
authors.forename: Wesley Leoricy
authors.initials: WL
authors.lastname: Marques
authors.affiliation: Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands.
authors.forename: Robert
authors.initials: R
authors.lastname: Mans
authors.affiliation: Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands.
authors.forename: Eko Roy
authors.initials: ER
authors.lastname: Marella
authors.affiliation: School of Food Engineering, University of Campinas, Campinas, SP 13083-862, Brazil.
authors.forename: Rosa Lorizolla
authors.initials: RL
authors.lastname: Cordeiro
authors.affiliation: Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands.
authors.forename: Marcel
authors.initials: M
authors.lastname: van den Broek
authors.affiliation: Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands.
authors.forename: Jean-Marc G
authors.initials: JG
authors.lastname: Daran
authors.affiliation: Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands.
authors.forename: Jack T
authors.initials: JT
authors.lastname: Pronk
authors.affiliation: School of Food Engineering, University of Campinas, Campinas, SP 13083-862, Brazil.
authors.forename: Andreas K
authors.initials: AK
authors.lastname: Gombert
authors.affiliation: Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands.
authors.forename: Antonius J A
authors.initials: AJ
authors.lastname: van Maris
mesh_terms: D001692:Biological Transport; D017353:Gene Deletion; D006868:Hydrolysis; D060847:Metabolic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D013395:Sucrose / Q000378:metabolism*
chemical_list: D013395:Sucrose
keywords: disaccharide; isomaltase; laboratory evolution; multiple gene deletion; real-time PCR; reverse engineering