A high-throughput screening method for improved R-2-(4-hydroxyphenoxy)propionic acid biosynthesis

R-2-(4-hydroxyphenoxy)propionic acid (R-HPPA) is a key intermediate of the enantiomerically pure phenoxypropionic acid herbicides. R-HPPA could be biosynthesized through selective introduction of a hydroxyl group (–OH) into the substrate R-2-phenoxypropionic acid (R-PPA) at C-4 position, facilitated by microorganisms with hydroxylases. In this study, an efficient high-throughput screening method for improved R-HPPA biosynthesis through microbial hydroxylation was developed. As a hydroxylated aromatic product, R-HPPA could be oxidized by oxidant potassium dichromate to form brown-colored quinone-type compound. The concentration of R-HPPA can be quantified according to the absorbance of the colored compound at a suitable wavelength of 570 nm; and the R-HPPA biosynthetic capability of microorganism strains could also be rapidly evaluated. After optimization of the assay conditions, the high-throughput screening method was successfully used in identification of Beauveria bassiana mutants with enhanced R-HPPA biosynthesis capacity. A positive mutant C-7 with high tolerance to 20 g/L R-PPA was rapidly selected from 1920 mutants. The biomass and R-HPPA titer were 12.5- and 38.19-fold higher compared with the original strain at 20 g/L R-PPA. This high-throughput screening method developed in this work could also be a potential tool for screening strains producing other important phenolic compounds.