Escherichia Coli Co-Culture System For Vanillyl Alcohol Biosynthesis

Vanillin is widely used in the food,beverage,cosmetics and pharmaceutical industries as a fragrance worldwide.At present,vanillin is mainly synthesized by chemical methods,but there are problems such as poor quality and serious pollution.The vanillin synthesized by biological methods has great potential in the market.However,vanillin is unstable and prone to redox.So,we choose the biosynthesis of vanillyl alcohol,the precursor of vanillin,in this study.In this study,our synthesis route was divided into 3,4-dihydroxybenzyl alcohol synthesis module and 3,4-DHBA conversion module.They were optimizated respectively.In the 3,4-DHBA synthesis module,through knocking out branch genes,enhancing the expression of key proteins in the shikimate pathway and screening the carbon source pathway,make the carbon flux doubled;by constructing the SAM cycle,the supply of methyl donors is effectively improved which led to a 1.58-fold increase of methylation capacity in the methylation module.But it occurs a problem of a large accumulation of intermediates in single-cell synthesis.To this end,we adopted the co-culture method,and we designed the carbon source separation and regulation strategy based on it.The upstream strains use glycerol as the sole carbon source,and the downstream strains use glucose as the sole carbon source,which is convenient for metabolic regulation and avoids the competition for carbon sources of strains.In order to increase the relationship between upstream and downstream strains,a growth-dependent system was constructed.The upstream strains formed AA-growth-deficient strains by knocking out the gene encoding shikimate dehydrogenase.This design not only further improved carbon flux,but also strengthen the stability of the co-culture system.Finally,in the shake flask level,the yield of vanillyl alcohol reached559.35±14.73 mg/L,which was 2.64 times higher than the synthesis in single cell.In fed-batch fermentation,stable production was achieved by adjusting the carbon source ratio,and the yield of vanillyl alcohol reached3890.23±87.89 mg/L.Compared with our previous results,the yield has increased by 16 times,which is the largest yield of de novo synthesis of vanillin.And this work has certain guiding significance for the industrial biosynthesis of vanillin.