Metabolic Engineering Of Saccharomyces Cerevisiae For Secretory Production Of Tocotrienols

Tocotrienols,as important components of vitamin E,are excellent antioxidants associated with diverse functions such as lowering cholesterol,neuroprotective and radioprotective activities,and anticancer activity,have wide applications in pharmaceutical,food,feed and cosmetics industries.Due to the lack of a chemical synthesis route,current market supply of tocotrienols depends on plant extraction,with issues of field occupancy and ecology damage.Recently,biosynthesis of tocotrienols using engineered microbial cell factories has emerged as an alternative production route.Aiming to explore efficient fermentative synthesis of tocotrienols,tocotrienols production in Saccharomyces cerevisiae was enhanced by pathway optimization targeting at precursor supply and redox balance,and the secretory production was explored via in situ extractive fermentation and overexpression of endogenous PDR proteins.First,the genes encoding regulatory factors such as MOT3 or ROX1 reported to de-repress enzymes in the mevalonate pathway and ergosterol synthesis were deleted from the previously constructed tocotrienols-producing S.cerevisiae strain YSM5,leading to strengthened supply of the MVA pathway-derived precursor for tocotrienols synthesis.Subsequently,GGPP synthesis was strengthened by overexpressing CrtE03M(encoding the C81T variant of GGPP synthase from Xanthophyllomyces dendrorhous).In the resulting strain,the tocotrienol yield reached 17.22 mg/g DCW,which was approximately 3.48-fold that of YSM5.Meanwhile,the squalene content dropped by 38.35%and the accumulation of the intermediates(MGGBQ and DMGGBQ)increased by 25.60%.Considering that overexpression of tHMG1 may disturb the cofactor balance in the engineered yeast,the mitochondrial NADH kinase POS5 was overexpressed to increase the intracellular NADPH/NADH ratio,which increased the tocotrienols yield to 17.92 mg/g DCW.After double deletion of MOT3 and ROXI,the resulting strain YVT17 produced tocotrienols with a yield of 19.24 mg/g DCW,which represents a 290%improvement over the starting strain YSM5.The lipophilic nature of tocotrienols leads to tendency of being stored in the cell membranes,which may exert stress on the yeast cells.If the product could be secreted to the culture medium,relief of cellular stress caused by its membrane storage could be expected,and meanwhile the downstream processing would be largely simplified.To test whether tocotrienols could be secreted into the organic phase during the production process,biphasic fermentation systems were established for strain YVT17 using different organic solvents as the extractant.When 5%(v/v)olive oil was added after 24 h cultivation,the maximum tocotrienol production of 76.6 mg/L was achieved,with 56.12%of the product secreted to the organic phase.In order to improve the transport efficiency of tocotrienols,we tentatively overexpressed the endogenous PDR transporters and their transcriptional activators in strain YVT17.By biphasic fermentation using olive oil as the extractant together with overexpression of endogenous PDR transporters PDR11 and YOL075C,the tocotrienol yield was improved to 25.57 mg/g DCW,and 73.66%was secreted into the organic phase.Besides tocotrienols as a mixture,single isomers of tocotrienols also have high market values.δ-tocotrienol is a potent radioprotectant and mitigator,and has functions in protecting primary neuronal cells from glutamate toxicity and reducing cholesterol levels.Considering its generally low content in plants and difficulty to separate from other tocotrienols,leading to high purification cost,construction of microbial cell factories exclusively producing δ-tocotrienol is a promising alternative route for production of this valuable product.To investigate the effect of different chassis organisms on tocotrienols production,the δ-tocotrienol synthetic pathway was assembled in S.cerevisiae strains BY4741,CEN.PK2-1 C,CEN.PK2-1D and Yarrowia lipolytica Polf.This is for the first time that synthesis of tocotrienols by engineered Yarrowia lipolytica was reported,albeit at a low yield(0.67 mg/L).Among all these strains,S.cerevisiae BY4741 gave the highest tocotrienols yield(7.75 mg/L),suggesting it as a suitable chassis for tocotrienols production.This result highlights the importance of screening suitable host strains in terpenoids synthesis.To promote tocotrienols production in S.cerevisiae BY4741,the precursor supply was enhanced by eliminating feedback inhibition of aromatic amino acids,downregulating competing pathways,optimizing the shikimate pathway,and enhancing tyrosine flux towards δ-tocotrienol production of the resulting strain produced 11.02 mg/L of δ-tocotrienol,together with 45.40 mg/L of MGGBQ as the direct precursor.The precursor supply was further strengthened by deletion of the gene encoding the regulatory factor ROX1 to relieve the restriction of the MVA pathway,increasing the copy number of CrtE03M to enhance the supply of GGPP,overexpressing the mitochondrial NADH kinase POS5 to improve the supply of cofactors,and knocking out the distal regulator DOS2 to improve the upstream metabolic flux.Subsequently,the copy numbers of the downstream rate-limiting enzymes TC and HPT were increased one-by-one to reconstruct the metabolic balance.The resulting strain YBVT15 produced 73.45 mg/L of δ-tocotrienol in shake flasks.Considering the solubility improvement of hydrophobic compounds by inclusion agents,2-hydroxypropyl-β-cyclodextrin(30 mM)was added during the cultivation process of strain.Together with overexpression of the Pleiotropic drug resistance transcription factor PDR1,the final δ-tocotrienol reached 243.57 mg/L(57.72 mg/g DCW)in shake-flask cultures.This process can achieve efficient production of single-isomer tocotrienol.This work does not only provide a potential strain for industrial bioproduction of tocotrienols,but also provides hints for yeast engineering towards secretory production of other terpenoids.