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

Wesley Leoricy Marques, Robert Mans, Eko Roy Marella, Rosa Lorizolla Cordeiro, Marcel van den Broek, Jean Marc G. Daran, Jack T. Pronk, Andreas K. Gombert, Antonius J.A. van Maris

Research output: Contribution to journalArticleScientificpeer-review

14 Citations (Scopus)
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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.

Original languageEnglish
Article numberfox006
Number of pages11
JournalFEMS Yeast Research
Volume17
Issue number1
DOIs
Publication statusPublished - 2017

Keywords

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

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