Research On Biosynthesis Pathway Of 2-hydroxyterephthalic Acid

High-performance p-phenylenebenzobisoxazole（PBO）fibers have excellent mechanical properties（stiffness,strength,and toughness）,high thermal stability,and light weight,and are widely used in automotive and aerospace composites,body armor and sports products.The hydroxyl-modified PBO（hydroxy-p-phenylenebenzbisoxazole,HPBO）fibers showed better photostability and interfacial shear strength.The monomer2-hydroxyterephthalic acid（2-HTA）of HPBO fiber is usually synthesized by chemical method,which has poor space selectivity and high energy consumption.This thesis aims to create a biological method to prepare 2-HTA.A new artificial biosynthesis pathway of 2-HTA is constructed,which uses grucose as raw material,generats chorismic acid by shikimate pathway,catalyzes by 3-hydroxybenzoic acid（3-HBA）synthase and hydroxybenzoic acid carboxylase to produce 2-HTA finally.Main research works are as follows:1.Discover the key enzyme in the pathway.Enzymatic Kolbe-Schmitt reaction converts phenolic compounds with bicarbonate/CO₂ to hydroxybenzoic acids.The project screened hydroxybenzoic acid carboxylase through the innovative application of enzymatic Kolbe-Schmitt reaction,and used 3-hydroxybenzoic acid as the substrate to carry out carboxylation at the para-position of the benzene carboxyl group,and biosynthesized 2-HTA.Three hydroxybenzoate（de）carboxylases from different microorganisms,2,3-Dihydroxybenzoic acid decarboxylase from Aspergillus oryzae（2,3-DHBD＿Ao）,2,3-Dihydroxybenzoic acid decarboxylase from Fusarium acuminatum（2,3-DHBD＿Fo）and salicylic acid decarboxylase from Trichosporon moniliiforme（SAD＿Tm）,were selected for expression in recombinant E.coli.Among them,2,3-DHBD＿Ao showed the highest activity in the whole-cell catalysis system using 3-HBA as substrate.The in vitro production of 2-HTA was108.97±2.21μg/L/mg protein.2.In order to further improve the enzyme activity,the enzyme was modified.The combination of 2,3-DHBD＿Ao crystal（7WKM）and small molecule 2-HTA was simulated,and the active center and binding pocket of the enzyme were predicted by Discovery Studio.The mutants of the active center F193 and binding pocket T62 was research.It was found that the aromic ring of F193 is necessary for catalytic activity.When F193was mutated to aliphatic amino acids 193I and 193L,the enzyme activity was completely lost.Although 193Y contained a benzene ring,its activity dropped to 43%of the wild type.Mutations T62G,T62 A,A62V increased the enzyme activity by 3.3,8.2,and 3.7 times respectively,which showed that reducing the steric hindrance of the binding pocket was an effective strategy to improve enzyme activity.The hydrophobicity of the binding pocket was analyzed and found to be positively correlated with enzyme activity,indicating that the enzyme catalytic activity was affected by both hydrophobicity and steric hindrance.Combined with the rationally designed F27G mutation,the double-site mutation F27G/T62A increased the production of 2-HTA by 24.7 times,and the in vitro production of 2-HTA reached to the 2.69±0.029 mg/L/mg protein.3.Optimize the enzymatic Kolbe-Schmitt reaction process to synthesize 2-HTA de novo.Using bicarbonate as the C1 source,0.5 M KHCO₃ can inhibit the growth of both E.coli and yeast strains.When the concentration of KHCO₃ was lower than 0.5 M,the carboxylation efficiency dropped significantly.Therefore,CO₂ was used as the C1 source for intracellular production of 2-HTA.The de novo 2-HTA synthesis pathway was constructed in Saccharomyces cerevisiae BY4741,and the exogenous 3-hydroxybenzoate synthase Hyg5,and the 2,3-DHBD＿Ao were expressed.The engineered strain was fermented and cultured in SC auxotrophic medium under CO₂ environment,and the 2-HTA titer was5.50±0.71μg/L at 48 h.4.Strategies such as increasing biomass,enhancing intracellular accumulation of intermediates and increasing carboxylase activity were adopted to increase yield of 2-HTA.The genes aro7 and trp3 for synthesis of the downstream aromatic amino acids（phenylalanine,tyrosine,tryptophan）can be knocked out to accumulate chorismate intracellularly.Therefore,Saccharomyces cerevisiae strain S228C（△ura3,△aro7 and△trp3）was used as the chassis strain to express 3-hydroxybenzoate synthase and carboxylase mutant 2,3-DHBD＿Ao^T62A.It synthesized 45.40±0.28μg/L 2-HTA in a CO₂ environment at 36 hours.A new pathway for 2-HTA biosynthesis was developed,which laid the foundation for the industrial production of 2-HTA,and proved the strong potential of hydroxybenzoate carboxylase in the production of terephthalic acid and its derivatives by fixing CO₂.