Metabolic Modification Of Saccharomyces Cerevisiae To Synthesize Pinene

Pinene is a plant-derived natural monoterpenoid compound,which has a wide range of applications in the chemical,pharmaceutical,perfume and other industries.In particular,a derivative of pinene,pinene dimer,has high energy value.It has great application prospects in high-fuel fields such as aerospace fields.With the development of synthetic biology,green biosynthesis of pinene is possible.Saccharomyces cerevisiae,as an excellent host for the synthesis of terpenes,has a strong ability in terms of precursor material supply,REDOX power and energy supply.In particular,S.cerevisiae has a good protein expression and modification system,which can express more complex enzymes to provide the basis for the synthesis of pinene dimers.Although the synthesis of pinene by S.cerevisiae had been reported,the main strategies currently focus on metabolic modification.Pinene synthase is a core functional enzyme.Here,the fusion expression protein of pinene synthase was constructed and the co-expression strategy of auxiliary proteins is adopted to improve the synthesis efficiency of pinene synthase and enhance the biosynthesis of pinene in S.cerevisiae.The following major research results are obtained:（i）Constructed the S.cerevisiae monoterpene production chassis strain Sc.MPS with enhanced MVA pathway:Increasing the overexpression of acetyl-Co A,t HMG1 and IDI1 to strengthen the mevalonate node of the MVA pathway and the restriction of IPP and DMAPP,Mutation of ERG20 to obtain ERG20^F96W-N127W,the original expression of these genes were integrated into the multi-copy site integration site of S.cerevisiae to obtain Sc.MPS;on this basis,the pinene synthase gene expression screen was screened,Pt1 and Pt30 from Pinus taeda were codon-optimized according to the codon preference of S.cerevisiae,and the pinene synthase expression plasmid was successfully constructed.Pt1 and Pt30 were truncated and point mutation screened,and the pinene synthase t Pt1 was determined to be better for pinene production,the output of pinene is 0.166 mg·L^-1.（ii）The fusion protein expression strategy was introduced.Fusion expression protein of ERG20^wwand t Pt1 were constructed and optimized:the（GS）₄ flexible linker was used to screen the fusion direction of ERG20^ww and t Pt1 fusion protein and determine the fusion sequence of ERG20^ww-t Pt1 is more optimal for pinene production;on the basis of this fusion sequence,the type of fusion linker was optimized,and the fusion protein of（G）₈ linker was determined to produce the best pinene yield;further the effect of the fusion expression of ERG20^ww and t Pt1genes on the production of pinene was investigated;after the optimization of the fusion protein,the output of pinene reached 8.05 mg·L^-1.（iii）Introducing the co-expression of auxiliary folding protein and fusion protein to improve the production capacity of the engineered S.cerevisiae strain pinene:combine nine auxiliary folding proteins from S.cerevisiae with the fusion expression proteins ERG20^ww-（GS）₄-t Pt1 and ERG20^ww-（G）₈-t Pt1 co-expression,found that Sil1p,Cpr5p,Jem1p and Scj1p have better results for the synthesis of pinene when the auxiliary folding protein is co-expressed with ERG20^ww-（GS）₄-t Pt1.Among them,the co-expression of Sil1p and ERG20^ww-（GS）₄-t Pt1increased the output of pinene by 16.5%to 8.25 mg·L^-1.However,when these nine auxiliary folding proteins are co-expressed with ERG20^ww-（G）₈-t Pt1,they generally show an inhibitory effect on pinene production.In this auxiliary folding protein,the performance of endoplasmic reticulum related auxiliary protein is better than that of cytoplasmic and mitochondrial origin.After the above research,the final yield of the pinene engineering yeast strain obtained was8.25 mg·L^-1,and the yield per cell reached the highest 1.32 mg·L^-1·OD^-1₆₀₀.