Screening of Fungi Isolates for C-4 Hydroxylation of R-2-Phenoxypropionic Acid Based on a Novel 96-Well Microplate Assay Method.

R-2-(4-Hydroxyphenoxy)propionic acid (R-HPPA) is a pivotal intermediate for the synthesis of aryloxyphenoxypropionate (APP) herbicide. To rapidly screen microbial isolates with the capacity of hydroxylating R-2-phenoxypropionic acid to R-HPPA from various environmental samples, a convenient and safe 96-well microplate assay method with sodium nitrite (NaNO2) as chromogenic reagent was proposed and optimized. The optimum assay conditions were as follows: the detection wavelength was 420 nm, the concentration of NaNO2 solution was 6.0 g/L, color reaction temperature was 60 °C, the pH of the NaNO2 solution was 2.4, and the reaction time was 40 min. With the aid of this method, screening for microorganisms with C-4-specific hydroxylation activity of R-PPA was conducted. As a result, 23 strains among 3744 single colonies isolated from various samples exhibited the hydroxylation activity. Among these strains, the highest bioconversion rate was achieved by Penicillium oxalicum A5 and Aspergillus versicolor A12, respectively. After 72-h cultivation in shake flask, their conversion rates of R-HPPA from 10 g/L R-PPA reached 21.18% and 40.24%, respectively. The established method was effective in rapid screening of microbes capable of biosynthesizing R-HPPA through hydroxylation of R-PPA, and the obtained two fungi species could be potentially used for R-HPPA production.

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.