Development of a dual temperature control system for isoprene biosynthesis in Saccharomyces cerevisiae

Abstract

Conflict between cell growth and product accumulation is frequently encountered in the biosynthesis of secondary metabolites. To address the growth-production conflict in yeast strains harboring the isoprene synthetic pathway in the mitochondria, the dynamic control of isoprene biosynthesis was explored. A dual temperature regulation system was developed through engineering and expression regulation of the transcriptional activator Gal4p. A cold-sensitive mutant, Gal4ep19, was created by directed evolution of Gal4p based on an internally developed growth-based high-throughput screening method and expressed under the heat-shock promoter P_SSA4 to control the expression of P_GAL-driven pathway genes in the mitochondria. Compared to the control strain with constitutively expressed wild-type Gal4p, the dual temperature regulation strategy led to 34.5% and 72% improvements in cell growth and isoprene production, respectively. This study reports the creation of the first cold-sensitive variants of Gal4p by directed evolution and provides a dual temperature control system for yeast engineering that may also be conducive to the biosynthesis of other high-value natural products.

Graphical abstract

Keywords

transcriptional activator / directed evolution / dynamic control / heat-shock / isoprene

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Acknowledgments

This work was financially supported by the National Key Research and Development Program of China (Grant Nos. 2018YFA0901800 and 2020YFA0908400), the National Natural Science Foundation of China (Grant No. 21776244), and Zhejiang Provincial Natural Science Foundation of China (Grant No. LZ20B060002).

Electronic Supplementary Material

Supplementary material is available in the online version of this article at https://dx.doi.org/10.1007/s11705-021-2088-0 and is accessible for authorized users.

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