High Efficiency Synthesis Of Myrcene,Ocimene And Bisabolol By Saccharomyces Cerevisiae

Terpenoids are the most abundant compounds in nature,which are distributed in various plants.Terpenoids are widely used in medicine,food,cosmetics,agriculture,etc.with great market demand.At present,commercial terpenoids are mainly extracted from plants,and a small part is synthesized by chemical methods.Limited by the low efficiency of plant extraction and low selectivity of chemical synthesis,it is difficult to meet the market demand.With the innovation of biotechnology and the vigorous development of synthetic biology,high hopes have been placed on efficient and environmentally friendly microbial fermentation.In this study,Saccharomyces cerevisiae was used as host to construct a cell factory for the efficient synthesis of myrcene,ocimene and bisabolol,providing a reference strategy for the synthesis of terpenoids in model microorganisms.The main research results are as follows:1)A platform strain for monoterpene production was constructed,and efficient synthesis of myrcene and ocimene was achieved.To construct a platform strain for monoterpene production in S.cerevisiae,we replaced the original promoter of ERG20 in the original strain SQ14 with two different weak promoters,P_ERG1 and P_HXT1,to reduce the loss of precursor GPP.To improve the efficiency of monoterpene synthesis,we screened monoterpene synthases from various sources,including myrcene synthase and ocimene synthase had the highest efficiency.To improve the availability of GPP,ERG20^F96W-N127W was expressed by fusion with monoterpene synthase,which shortened the spatial distance between the substrate and the enzyme and greatly increased the metabolic flux.The optimization of the two-phase fermentation process is beneficial to the synthesis of the product.We optimized the extraction solvent,addition amount,addition time and other parameters of the two-phase fermentation on the shake flask,and the addition of 15%dodecane at 12 h was the most conducive to the accumulation of myrcene.The titer of myrcene and ocimene reached 8.12 mg·L^-1 and 34.56mg·L^-1,respectively.2)An engineered strain of S.cerevisiae for high-efficiency synthesis of（–）-α-bisabolol was constructed by systematic metabolic engineering.ERG9,a key competitive pathway of（–）-α-bisabolol synthesis,was weakened,and the fusion protein of Erg20 and Mr BBS was constructed and optimized.The optimal linker form was determined,and the optimal strength promoter P_GAL7 was screened for the fusion protein.Overexpression of ERG20 and ERG10,two possible rate-limiting genes in the MVA pathway,showed little effect on the synthesis of（–）-α-bisabolol,but the knockout of global transcription factor ROX1 significantly increased the titer of（–）-α-bisabolol,indicating that global regulation is more effective than local adjustment.The potential terpene-transport-related genes reported in the literature were knocked out,and the genes PDR15 and PDR3 that significantly changed the ratio of intracellular and extracellular（–）-α-bisabolol were overexpressed,and the overexpression of PDR15 significantly increased the extracellular（–）-α-bisabolol content.In addition,we overexpressed genes associated with ATP/NADPH synthesis,the key cofactors in the MVA pathway,but decreased production,suggesting that the cofactors may not be a limiting factor in production of（–）-α-bisabolol.3)The fermentation process of the strain was optimized at the level of shake flask and 5 L bioreactor,respectively.Through the initial glucose concentration,feeding strategy,and the addition of calcium carbonate,the titer of the strain in the shake flask was improved.After that,we made further fermentation optimizations in the 5 L bioreactor.Firstly,we optimized the initial glucose concentration and showed that an initial glucose concentration of 30 g·L^-1 could promote the accumulation of（–）-α-bisabolol while maintaining a high biomass of the strain.Secondly,we added 10 g·L^-1 calcium carbonate to the initial medium,maintained a relatively constant p H of the medium in the bioreactor.The results showed that adding glucose at 0-60 h and absolute ethanol after 60 h was the optimal fed-feeding process,and the final total titer of（–）-α-bisabolol was improved to 7.02 g·L^-1,which is the highest titer of（–）-α-bisabolol synthesized in yeast.