Improvement of 2,3-butanediol production by dCas9 gene expression system in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae has been widely used in bioproduction. To produce a target product other than ethanol, ethanol production must be decreased to enhance target production. An ethanol non-producing yeast strain was previously constructed by knocking out pyruvate decarboxylase (PDC) genes in the ethanol synthetic pathway. However, glucose uptake by the ethanol-non-producing yeast strain was significantly decreased. In this study, dead Cas9 (dCas9) was used to reduce ethanol synthesis during 2,3-butanediol production without reduction of glucose. The binding site of guide RNA used to effectively suppress PDC1 promoter-driven red fluorescent protein expression by dCas9 was identified and applied to control PDC1 expression. The production of 2,3-butanediol rather than ethanol was improved in repetitive test tube culture. Additionally, ethanol production was decreased and 2,3-butanediol production was increased in the strain expressing dCas9 targeting the PDC1 promoter in the third round of cultivation, compared with the control strain.

Keywords: 2,3-Butanediol production; CRISPR interference; Metabolic engineering; Saccharomyces cerevisiae; dCas9 expression system.