Biosynthesis Of 4-Hydroxyphenylglycine(HPG) Monomer And HPG-Containing Biopolyesters

L-4-hydroxyphenylglycine(L-HPG)is a non-protein-derived amino acid,which is an intermediate in the synthesis of some new anticancer drugs and non-ribosomal peptide antibiotics(such as vancomycin),and has important application prospects in the medical field.At present,L-HPG is mainly synthesized by chemical methods,and mature biosynthesis methods have not been realized.In this study,the biosynthesis method of L-tyrosine to L-HPG was constructed from engineering Escherichia coli.In addition,the hydroxyl and carboxyl structures of L-HPG have potential application prospects of aromatic polyester monomers.Therefore,this study explored the biosynthesis of novel aromatic polyesters using L-HPG as a monomer.The main results of this study are as follows:(1)In this study,genes of 4-hydroxyphenylpyruvate dioxygenase(Hmas),4-hydroxyamygdalate oxidase(Hmo)and 4-hydroxyphenylglycine transferase(Hpg T)from Streptomyces coelicolor were recombined into Escherichia coli chassis cells,the engineered Escherichia coli producing L-HPG was constructed.(2)Starting from the construction of L-HPG-producing engineering E.coli,this study further recombined the genes of 2-hydroxyisohexanoate transferase(Had A)from Clostridioides difficile ATCC 9689 and PHA(PHA_ps6-19)type II synthetase from Pseudomonas sp.MBEL 6-19 into the engineering bacteria,and the polyester preparation engineering bacteria containing L-HPG monomer were constructed.(3)In this experiment,starting from the constructed L-HPG-producing engineering strain,the fermentation process of L-HPG was systematically studied,and the qualitative and quantitative analysis of biosynthetic L-HPG was carried out.Compared with the standard product,the results of liquid mass and high performance liquid phase confirmed that the engineered bacteria could synthesize L-HPG with L-tyrosine as the substrate.The results of quantitative analysis confirmed that the synthesized target product L-HPG could reach 0.47 g/L at the level of shake flask.(4)In this study,the fermentation and separation analysis of L-HPG-containing aromatic polyesters were carried out based on the constructed engineering bacteria containing polyester biosynthesis pathway.Although the final product was extracted from the fermentation at the shake flask level,the qualitative analysis of the product(NMR)showed that the synthesized product was complex,which needs to be discussed and analyzed,suggesting that the transferase used by the original engineering bacteria has low substrate specificity for L-HPG and cannot convert L-HPG into coenzyme-based active monomers for polyester synthesis.(5)In this study,the transferase gene in the polyester synthesis engineering bacteria was further replaced,but the fermented product still did not have the product we expected.The results confirmed that the polyester synthesis pathway screened in this study could not successfully synthesize aromatic polyesters containing HPG monomers,suggesting that the transferases and polyester synthases used need to be further optimized for the biosynthesis of novel aromatic polyesters.