Improved Linalool Production In Saccharomyces Cerevisiae By Comprehensive Control

Linalool as an acyclic monoterpene tertiary alcohol is widely found in plant essential oil Because of its distinctive fragrance and various biological functions,linaloolhas become an important feedstock for food,pharmaceuticals,and cosmetics industries.Currently,linalool is mainly extracted from plant or produced by chemical synthesis.However,Extracting linalool from plant suffered from Low yield and complex process of separation and purification,which can not meet the growing demand of our society.Linalool synthesized by chemical method is the mixture of the mesomer and raceme with no configurational selectivity.In addition,the process of chemical synthesis of lianlool is complex and the cost is relatively high.In recent years,with the development of synthetic biology in recent years,it has become a trend for Metabolic engineering of microorganisms to produce natural compounds.In this study,Saccharomyces cerevisiae was used as the chassis cell for linalool production using protein engineering and metabolic engineering method.Firstly,the strain YLin-01 with sufficient precursor supply was constructed by overexpressing the rate limiting enzyme of mevalonate(MVA)pathway and ERG20 mutant(ERG20F96W/N127W)with high Geranyl diphosphate(GPP)synthesis efficiency to improve precursor supply.Different Linalool synthases were expressed in the strain YLin-01,respectivelyathe relatively high activity of linalool synthase(t67OMcLIS)was obtained andthe resultingstrain YLin-03 accumulated 3.32 mg/L of linaloolIn order to further improve the supply of precursor,BY4741-C04,which overexpressed the whole MVA pathway,was used as the starting strain.With sufficient precusor,the efficiency of linalool biosynthesis largely depends on the c activity of linalool synthase Based on the competition of linalool and lycopene for the common precusor GPP,a lycopene-indicated high-throughput screening method was established for directed evolution of t670McLIS.Finally,a mutant t670McLISE343D/E352H with improved activity was obtained The YLin-05 strain carrying the t670McLISE343D/E352H produced 53.14 mg/L of linalool exhibited a 52.7%increase compared with YLin-04.However,The biomass of YLin-05 was decreased by 36.4%compared with YLin-03,which might be resulted from the metabolic burden by overexpression of the whole MVA pathway genes.To reduce the cellular metabolic stress caused by multi-genes overexpression and promote conversion of toxic intermediates,only several key enzymes of MVA pathway was overexpressed,meanwhile combinatorial modulation oft670McLISE343D/E352H and ERG20F96W/N127W was adopted.Firstly,the expression level of linalool synthase was increased by introducing a N-terminal SKIK tag,which improve the linalool production by by 3.4-fold.Then,the t67OMcLISE343D/E352H and ERG20F96W/N127W proteins were assembled via a pair of short peptide tags(RIAD and RIDD),leading to the production of linalool increased by 42.1%.Further different promoters were screened to downregulate the endogenous FPP synthase(ERG20)in S.cerevisiae,the resulting strain YLin-12 with no influence of biomass,accumulated 80.9 mg/L of linalool,which was the highest yield reported in S.cerevisiae.In this study,an efficient linalool-producing S.cerevisiae was constructed via adopting a series of protein engineering and metabolic engineering strategies,Which provide a reference for the green synthesis of linalool in future and meanwhile provide methodological method for biosynthesis of other monoterpenes.