Metabolic Engineering Of Saccharomyces Cerevisiae For Terpenoids Production

Terpenoids have high economic and application value in many aspects,such as food,pharmaceuticals and chemicals.It is difficult to meet the growing market demand by extraction from natural sources and chemical synthesis.The production of terpenoids by microbial cell factories is considered a green and economical method.Saccharomyces cerevisiae has advantages of high biological safety,easy genetic manipulation.Thus,the use of S.cerevisiae and cheap carbon sources is becoming a valuable route for the synthesis of terpenoids.At present,there are many strategies for the heterologous synthesis of terpenoids in yeast,including introduction and overexpression of heterologous pathway enzymes,increasing the supply of precursors,down-regulating or deleting competing pathways and screening suitable fermentation substrates to reduce costs.Lipophilic terpenoids are generally considered to accumulate and encapsulate in lipid droplets(LD).However,the metabolic synthesis of each component in LD and the synthesis of terpenoids share a variety of precursor,ATP and NADPH,which complicate the entire metabolic network.The production of terpenoids in yeast is affected by many factors such as the regulation of metabolic networks and the toxic effects of metabolites.Therefore,it is important to increase terpenoids production through altering yeast lipid metabolism.S.cerevisiae Invertase(Sc In V)encoded by SUC2 is the key enzyme for hydrolysis of sucrose and participation in a variety of biochemical reactions in S.cerevisiae.Sucrose fermentation helps to improve the production of high-value products.Therefore,it is necessary to research the function and activity of invertase mutants.In this study,CEN.PK 113-5D strain was used as the host strain.We characterized the effect of flux redirection of lipid metabolism pathways on??carotene production and?-humulene production in yeast.In addition,we also reduced the Sc In V mutant enzyme activity by site-directed mutation,and research the effects of different carbon sources on?-carotene accumulation and other characteristics.It provides a certain reference for the use of cheaper molasses to produce high-value compounds in S.cerevisiae.The main research results are as follows:1.To characterize the ability of S.cerevisiae to accumulate different terpenoids,the heterologous pathway enzymes of?-carotene and?-humulene were introduced into yeast cells,respectively.By integrating crt YB,crt I,crt E gene and an N?terminal truncated version of HMG1(t HMG1)in the yeast genome,and the engineered YZ03 strain could produce??carotene at 3.67mg/g DCW.By integrating ZSS1 gene in the yeast genome and integrating an additional copy of ERG20 with strong constitutive promoters in chromosome XII,and replacing the ERG9 promoter with ERG1 promoter,the resulted ZH04strain could produce?-humulene at 93.94 mg/L.These results indicate that we have successfully constructed an engineered strain that produces?-carotene and?-humulene in S.cerevisiae.2.Various functions of cells are related to the regulation of lipid metabolism network.We studied the effect of flux redirection of lipid metabolism pathways on different terpenoids production in yeast.We overexpressed ARE1 and ARE2 by integrating an additional copy of ARE1 and ARE2 with strong constitutive promoters in chromosome XII,and produced5.67 mg/g DCW?-carotene,which is 54%higher compared with that of the reference strain YZ03.Deletion of phosphatidate phosphatase genes(PAH1,DPP1 and LPP1)also increased??carotene yield by twofold.Combining these two strategies resulted in a 2.4?fold improvement in??carotene production compared with the starting strain.These results demonstrated that regulating lipid metabolism pathways is important for??carotene accumulation in S.cerevisiae.However,overexpression of ARE1 or deletion of the phosphatidate phosphatase genes reduced the production of?-humulene.This indicates that for different terpenoids,the relationship between their accumulation and lipid metabolism network is very complicated in yeast.Although the specific mechanism is not clear,this work also shed insights to study the relationship between altered lipid metabolism networks and terpenoids accumulation.3.S.cerevisiae contains invertase,which can utilize sucrose for fermentation.We researched the effects of different invertase activities under sucrose conditions on?-carotene accumulation and other characteristics.We designed standardized vectors with different tags and compatible with Golden Gate cloning,and constructed several protein expression plasmids with different tags.It enables to complete the site-directed mutagenesis of Sc In V quickly and efficiently,which accelerates the expression of the mutant proteins.Under high sucrose conditions,SUC2^Q148A and SUC2^Q201V mutants have lower invertase activity and enhanced fermentation capacity,which provides a certain reference for strain screening.