摘要
Vanillin,a valuable aromatic compound,is widely used in the food and medical industries.Vanillin production using cell factory is considered a promising alternative to petro-based synthesis,however,formation of by-products,insufficient supply of precursors and cofactors have caused the significant challenge to high-level production.In this study,we focused on the de novo synthesis of vanillin in E.coli through preventing the accumulation of by-products,optimizing the supply of L-tyrosine and cofactors,and designing a co-culture system.To reduce vanillic acid accumulation,a carboxylic acid reductase(MmCAR)and its reactivator phos-phopantetheinyl transferase(SFP)was screened,resulting in 88.1%decrease of vanillic acid.Deleting alcohol dehydrogenases(yahK yjgB and yqhD)efficiently inhibited the formation of vanillyl alcohol,resulting in a 4.25-fold increase in vanillin production.Deleting tyrR and pheA,and overexpressing aroG^(D146N )and tyrA^(M53I,A354V) to alleviate their feedback inhibitions further enhanced the titer of vanillin 45.96%.Increasing NADPH regenera-tion by overexpressing pos5(encoding NADH kinase)and promoting S-adenosyl-l-methionine(SAM)regenera-tion by expressing mtn(encoding 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase)and luxS(encoding S-ribosyl-homocysteine lyase)resulted in the titer of vanillin to 279.17 mg/L.Finally,the use of a E.coli co-culture system achieved the production of 465.81 mg/L vanillin,the best reported titer in E.coli.These results provide a new opportunity for de novo production of vanillin from glucose.
基金
supported by National Natural Science Foundation of China(Grant No.22478255).
