Microbial synthesis of m-tyrosine via whole-cell biocatalysis

Meta-tyrosine (m-tyrosine), a nonproteinogenic amino acid, has shown significant potential for applications as an herbicide in agriculture and for various medical uses. However, the natural abundance of m-tyrosine is very low, limiting its widespread use. In this study, we successfully achieved microbial production of m-tyrosine by establishing the in vivo enzyme activity of phenylalanine 3-hydroxylase (PacX from Streptomyces coeruleoribudus) in E. coli, which catalyzes the meta-hydroxylation of phenylalanine to produce m-tyrosine. Remarkably, PacX is capable of utilizing the native E. coli cofactor tetrahydromonapterin (MH4) for its hydroxylation activity. The integration of a non-native MH4 regeneration system significantly improved the bioconversion efficiency, resulting in the accumulation of m-tyrosine at a concentration of up to 368 mg/L. Additionally, we attempted to modify a well-characterized phenylalanine 4-hydroxylase (P4H) from Xanthomonas campestris to alter its regioselectivity through protein engineering. Remarkably, a double mutant (F184C/G199T) successfully shifted the enzyme's hydroxylation specificity from the para- to the meta-position, demonstrating the feasibility of altering the regioselectivity of aromatic amino acid hydroxylases (AAAHs). To the best of our knowledge, this is the first report of microbial production of m-tyrosine through whole-cell biocatalysis.