Design And Construction Of Saccharomyces Cerevisiae For Production Of Protocatechuic Acid And Vanillin From Lignin

Lignocellulose is the most abundant renewable resource in nature.Conversion of lignocellulose can reduce the use of oil and coal,which is important to “carbon neutrality”.Lignin is mainly a three-dimensional reticulated polymer with phenylpropanoid units as the main structure,which is the most abundant renewable aromatic resource in nature.Lignin,as one of the three major components of lignocellulose,has not yet been reasonably utilized.Conversion of lignin to synthesize valuable aromatic compounds,such as protocatechuic acid and vanillin can effectively utilize the aromatic ring structure of lignin,promote lignin valorizations,and reduce the dependence on fossil resources.Here,a “biological funnel” pathway was designed and constructed for the synthesis of protocatechuic acid in Saccharomyces cerevisiae using lignin-derived aromatics as substrates.The S.cerevisiae chassis was optimized by knocking out key gene to reduce the branched metabolism of substrates and intermediates in the“biological funnel” pathway and promoted the aromatic acid synthesis.Increasing the expression of Pob A improved the hydroxylation capacity.In addition,introduction of different demethylase and regulate tetrahydrofolate metabolism to enhance transfer of methyl group,which improved the vanillic acid demethylation.These strategies increased the conversion rate of protocatechuic acid synthesis to 66.7% from pcoumaric acid and 75.5% from ferulic acid.In further,810 mg/L of protocatechuic acid was obtained from lignin hydrolysate with the fermentation process optimization.A synthetic “biological funnel” pathway of vanillin synthesis from lignin monolignol such as ferulic acid,p-coumaric acid and p-hydroxybenzaldehyde was designed and constructed by introducing the non-β-oxidation,hydroxylation,methylation through enzyme screening and identifying.To reduce the metabolism of vanillin in S.cerevisiae,the genes ADH6,ADH7,BDH1,and BDH2 were knocked out to reduce the production of vanillyl alcohol.In further,the vanillin metabolism was reduced by mining and deleting ARI1,GRE2,and HFD1 genes,and more than 80% of vanillin remained after 48 h culture with initial 200 mg/L vanillin.The hydroxylation was enhanced by fusion protein strategy.Regulation of the S-adenosylmethionine synthesis and regeneration by overexpressing the MET13 scat,MET6,and SAH1 genes,couple with improving the expression of methylation were further applied to enhance the methylation.This“biological funnel”pathway was optimized through above strategies to enhance the conversion rate,which 84.5% and 32% conversion rate of vanillin synthesis from ferulic acid and p-coumaric acid were achieved,respectively.Finally,1.35 mmol/L vanillin were produced from lignin stream in the flask fermentation.This study demonstrated a novel and green strategy for the improvement of lignin valorization,which provided a new route for the biosynthesis of aromatic compounds such as protocatechuic acid and vanillin.