Heterologous Biosynthesis Of Vitamin E(Tocotrienols)in Saccharomyces Cerevisiae

Vitamin E is an important fat-soluble compound,which refers to the mixture of four tocopherols and four tocotrienols.It is an essential vitamin for maintaining normal metabolism and function of the body,however we cannot synthesize Vitamin E and must obtain it from food.It has the functions of scavenging free radicals,anti-cancer,anti-cardiovascular disease,anti-aging and so on.Compared to tocopherols,tocotrienols have more outstanding functions of reducing cholesterol,antioxidant,anti-cancer,anti-inflammation and heart protection.Natural vitamin E is only found in photosynthetic organisms,like algae,cyanobacteria and green plants.The natural vitamin E in the market is mainly extracted from deodorized distillate of vegetable oil,but it can not meet the growing market due to its low content,limited source and difficulty in separation.Chemically synthesized vitamin E,which is racemic a-tocopherol,has low biological activity and is mainly used for feed additives.To provide a possible alternative route for production of tocotrienols,this study constructed a tocotrienol synthetic pathway in Saccharomyces cerevisiae based on the concept of synthetic biology,by extending the endogenous shikimate pathway and mevalonate pathway(MVA)with the biosynthetic pathway of vitamin E in photosynthetic biosynthesis.Firstly,a series of key pathway genes for the synthesis of vitamin E were cloned from photosynthetic organisms such as Arabidopsis thaliana,Cyanobacteria,Nicotiana tabacum and other photosynthetic organisms.EGFP was used as a reporter to initially screen for enzymes that were successfully expressed in S.cerevisiae.Combining codon optimization and product detection,five key enzymes with expected catalytic activity were obtained,namely HPPD,syHPT,MPBQMT,TC and y-TMT.These pathway genes were then gradually integrated into the S.cerevisiae strain YS40 constructed by overexpressing tHMG1 and CrtE and knocking out GAL80,using the pUMRI assembly vector constructed by our research group.The resulting strain YS-16 produced 172?g/g DCW of y-tocotrienol and 71.3?g/g DCW of a-tocotrienol.To further improve the production of tocotrienols,we predicted and exculated the transit peptides of the three downstream enzymes MPBQMT,TC and y-TMT from A.thaliana to improve their expression level and catalytic efficiency,resulting in the optimized strain YS-16C,which increased the yield of total tocotrienols by 1.59 times,with 395.2?g/g DCW of y-tocotrienol and 236.1?g/g DCW of a-tocotrienol.We further overexpressed the rate-limiting enzymes syHPT,TC and y-TMT to construct a high-yield strain YS-356C,which increased the total tocotrienols yield by 7.54 times compared to the original strain YS-16,reaching 2.09 mg/g DCW,of which the yield of y-tocotrienol and a-tocotrienol was 1407.9?g/g DCW and 677.4?g/g DCW.At the same time,combined with the optimization of the upstream shikimate pathway and the MVA pathway,and increasing the supply of endogenous SAM,the total production of tocotrienols is further improved through comprehensive regulation.This is the first time to achieve heterologous synthesis of y-tocotrienol and a-tocotrienol in non-photosynthetic organisms,and to synthesize natural vitamin E by bio-fermentation,which provides new ideas and methods for the production of natural vitamin E.