Development of a temperature‐responsive yeast cell factory using engineered Gal4 as a protein switch

Abstract

Conflict between cell growth and product accumulation is frequently encountered in biosynthesis of secondary metabolites. Herein, a temperature-dependent dynamic control strategy was developed by modifying the GAL regulation system to facilitate two-stage fermentation in yeast. A temperature-sensitive Gal4 mutant Gal4M9 was created by directed evolution, and used as a protein switch in ΔGAL80 yeast. After EGFP-reported validation of its temperature-responsive induction capability, the sensitivity and stringency of this system in multi-gene pathway regulation was tested, using lycopene as an example product. When Gal4M9 was used to control the expression of P -driven pathway genes, growth and production was successfully decoupled upon temperature shift during fermentation, accumulating 44% higher biomass and 177% more lycopene than the control strain with wild-type Gal4. This is the first example of adopting temperature as an input signal for metabolic pathway regulation in yeast cell factories.

Fermentative production of Vitamin E tocotrienols in Saccharomyces cerevisiae under cold-shock-triggered temperature control

“…As reported for carotenoids biosynthesis, accumulation of lipophilic products in cell membrane often leads to growth inhibition 30 . Although the glucose-responsive regulation system based on GAL80 deletion 31,63 was adopted, the slow growth and low final biomass suggested failure of this system to relieve metabolic burden of these cells, possibly due to the leaky expression of the heterologous pathway at low glucose concentrations 32 . Accumulation of the target products prior to the planned switch-on time may impair cell growth.…”

“…To weaken P GAL1 while maintaining its response to Gal4 binding, truncated P GAL1 variants were created by changing the number of Gal4 binding sites in UAS G , generating P GAL1 -2 with 3 sites and P GAL1 -3 with 2 sites 54 . Examination of the promoters in the lycopene-producing yeast Ylyc-TS0 32 finalized P GAL1 -3 as the one with appropriate strength to control Gal4 expression, and 5 h of 24°C incubation was found sufficient to deliver temperature control ( Supplementary Note 1). Introduction of the cold-shock-triggered temperature control system into the tocotrienols high-producing strain YS-M5 generated strain YS-M5GP3 (YS-M5, ΔGAL4::HIS3, ΔDPP1:: P GAL1-3 -GAL4, ΔGAL80::P GAL4 -GAL4M9).…”

“…In order to achieve high production of tocotrienols, the conflict between product accumulation and cell growth which is commonly encountered in biosynthesis 30 should also be addressed. In previous efforts towards high production of carotenoids, we have developed glucose-responsive and temperature-responsive regulation systems to separate the production stage from the growth stage 31,32 . In particular, the temperature-responsive control system based on Gal4 engineering could lead to tight regulation of two-stage fermentation 32,33 .…”

“…In previous efforts towards high production of carotenoids, we have developed glucose-responsive and temperature-responsive regulation systems to separate the production stage from the growth stage 31,32 . In particular, the temperature-responsive control system based on Gal4 engineering could lead to tight regulation of two-stage fermentation 32,33 . However, trade-off between activity and temperature sensitivity occurred during directed evolution of Gal4, and the lower activity of the temperature-sensitive mutant Gal4M9 would limit the production of target metabolite.…”

Microbial Cell Factory for Efficiently Synthesizing Plant Natural Products via Optimizing the Location and Adaptation of Pathway on Genome Scale

“…The marker was removed through low-frequency mitotic recombination and colony counterselection. The improved decentralized assembly strategy was applied to the synthesis of isoprenoids (Ye et al, 2017) and lycopene (Zhou et al, 2018).…”