Construction And Optimization Of Cu²⁺ Response GAL Regulation System In Saccharomyces Cerevisiae

With its excellent comprehensive characteristics,Saccharomyces cerevisiae is an ideal chassis cell for the biosynthesis of high value-added compounds,and plays an essential role in the field of synthetic biology.However,the mutual interference and imbalance between product accumulation and cell growth is a common problem in the heterogeneous biosynthesis of natural products.Therefore,developing a simple,efficient,and strict regulation system is of great significance to realize the controllable synthesis of target compounds via dynamically controlling the expression of exogenous multiple genes.In this study,Combined galactose regulatory system(GAL)with Cu2+responsive promoters in S.cerevisiae,a low-cost and efficient Cu2+responsive GAL regulatory system(CuIGR)is constructed by protein expression optimization and engineering modification,which provide a reference for the biosynthesis of natural products.First,the endogenous GAL4 promoter was replaced with the copper-inducible PCUP1 using the CRISPR/Cas9 editing system,leading to the expression of Gal4 was induced by Cu2+.Meanwhile,the endogenous GAL80 promoter was replaced with the copper-inhibited PcTR3,resulting in the expression of Gal80 be inhibited by Cu2+.The modified GAL regulatory system get rid of the dependence on the expensive inducer galactose,and is only regulated by the cheap inducer Cu2+.However,due to strong expression of PCTR3-driven Gal80 in the absence of Cu2+,and long half-life of Gal80,even if addition of Cu2+,the accumulated Gal80 in the beginning could still inhibits the activity of Gal4,resulting in low inducible efficiency of the regulatory system.Therefore,we optimized the expression of GAL80 by shortening the half-life of Gal80.Four different half-life of Gal80(K3K15-Gal80,KN113-Gal80,KN4-Gal80 and K15-Gal80)was constructed by using with four different tags for degradation.The CuIGR4 system which the Gal80 was modified by K15 degradation tag showed the lowest leakage expression and highest induction efficiency.When the lycopene pathway genes in S.cerevisiae was regulated by the CuIGR4 system,the titer of total carotenoids was only 3.03 mg/L without Cu2+addition,While induction with 50 μM Cu2+after 22 h,the yield of total carotenoids reached 107.45 mg/L,Which increased 35.5 folds compared with the yied without induction.In addition,we also optimized the inducer concentration and induction time.Addition of 20 μM Cu2+at 22 h was the optimal condition for induction.In order to fuher improve the inducible efficiency of the CuIGR4 system,increasing the activity of Gal4 might be an alternatice method.In this study,based on the lycopene-indicated high-throughput screening method,directed evolution of Gal4 was performed.Six mutants of Gal4 with increased activity(Gal4S6P,Gal4T406A,Gal41407V,Gal4V413A,Gal4K459R and Gal4V586A)were successfully screened.Subsequently,the best Gal4 mutant(Gal4T406A/V413A)was obtained through combinatorial mutagenesis,leading to the lycopene production increased about 48%.In the further study,the mutant could be applied in the CuIGR4 system,to improve its inducible efficiency.