High-yielding Protocatechuic Acid And Cis,Cis-muconic Acid Synthesis From Renewable Feedstocks By Biocatalysis

Protocatechuic acid(PCA)and cis,cis-muconic acid(ccMA)are widely used in various industries.PCA has excellent biological activities and pharmacological effects such as antioxidant,antibacterial,antiaging,antitumor and antiinflammatory,and is used in pharmaceutical or functional foods.Cis,cis-muconic acid is a precursor to adipic acid for nylon-66 synthesis.These chemicals are traditionally extracted from plants or synthesized from petroleum-based feedstocks.The biocatalysis approach to convert renewable raw materials into valuable fine chemical products is considered as an economical and environmentally friendly alternative to traditional chemical methods.In this study,we aimed to create a multi-enzyme cascade that can efficiently convert L-tyrosine to PCA,thus obtaining a high-yield recombinant Escherichia coli(E.coli)strain.We developed two artificial enzyme cascade catalytic pathways for converting L-tyrosine into PCA.The first route comprises L-amino acid transaminase(LAAD),E.coli hydroxylation enzyme(HpaBC),hydroxymandelate synthase(HmaS),hydroxymandelate oxidase(HMO),benzoylformate decarboxylase(BFD)and aldehyde dehydrogenase(ALDH).Since the intermediate 4-hydroxybenzoate(HBA)was accumulated in the first artificial pathway,we further attempted a second pathway to convert HBA into PCA using HBA hydroxylase(PobA).When the biocatalytic system was added with 25 mM L-tyrosine,the concentrations of PCA reached 23.33 mM with 93.3%conversion.In addition,we also developed an artificial pathway for ccMA production from lignin-derived isoeugenol.In order to eliminate the bottleneck of isoeugenol as a competitive inhibitor to prevent the demethylation of vanillic acid(VAN)to PCA,a two-step catalytic method was used.The first step enzyme module contains aromatic dioxygenase(ADO)and aldehyde dehydrogenase(ALDH)could efficiently transform isoeugenol to VAN.Then the second step module introduces three enzymes that are VAN O-demethylase(VanAB),gallat decarboxylase(GDC)and catechol dioxygenase(CatA).By optimization of the pH conditions and the feeding strategy,VAN was completely consumed and 43.8 mM ccMA was synthesized from 50 mM VAN with 87.6%conversion rate(without dilution factor).In summary,we have designed and validated multi-enzyme cascades to convert renewable feedstocks to PCA and ccMA,and the recombinant E.coli strains with high yield of PCA or ccMA were established,respectively.It provides an alternative method for synthesis of PCA and other high value-added products.In the future,we will attempt to integrate these enzymatic modules into the E.coli chromosome to avoid the use of antibiotics for maintaining the plasmids,fine-tune the expression level of individual gene components and to develop even more robust whole-cell biocatalysts that are more suitable for industrial applications for PCA and ccMA productions.