Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism

Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism

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)

2017

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.

Disaccharide
Isomaltase
Laboratory evolution
Multiple gene deletion
Real-time PCR
Reverse engineering

http://resolver.tudelft.nl/uuid:da98a321-fc15-4df8-bd83-e7d16ea1420a

https://doi.org/10.1093/femsyr/fox006

1567-1356

FEMS Yeast Research, 17 (1)

Institutional Repository

journal article

© 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