Construction of a Convenient Screening Method for P-Hydroxybenzoate Hydroxylase To Enable Efficient Gallic Acid and Pyrogallol Biosynthesis

Abstract

BackgroundGallic acid (GA) and pyrogallol are phenolic hydroxyl compounds and have diverse biological activities. Microbial-based biosynthesis of GA and pyrogallol has been emerged as an ecofriendly method to replace the traditional chemical synthesis. In GA and pyrogallol biosynthetic pathways, the low hydroxylation activity of p-hydroxybenzoate hydroxylase (PobA) towards 3,4-dihydroxybenzoic acid (3,4-DHBA) limited the high-level biosynthesis of GA and pyrogallol.ResultsThis work reported a high active PobA mutant (Y385F/T294A/V349A PobA) towards 3,4-DHBA. This mutant was screened out from a PobA random mutagenesis library through a novel naked eye visual screening method. In vitro enzyme assay showed this mutant has a kcat/Km of 0.059 μM-1s-1 towards 3,4-DHBA, which was 4.92-fold higher than the reported mutant (Y385F/T294A PobA). Molecular docking simulation provided the mechanism explanation of the high activity. Expression of this mutant in E. coli BW25113 (F’) can generate 830 ± 33 mg/L GA from 1000 mg/L 3,4-DHBA. After that, we utilized this mutant to assemble a de novo GA biosynthetic pathway. Subsequently, this pathway was introduced into a 3,4-DHBA-producing strain (E. coli BW25113 (F’)ΔaroE) to achieve 301 ± 15 mg/L GA production from simple carbon sources. Similarly, assembling this mutant into a de novo pyrogallol biosynthetic pathway enabled 129 ± 15 mg/L pyrogallol production.ConclusionsThis work established an efficient screening method and generated a high active PobA mutant. Assembling this mutant into GA and pyrogallol biosynthetic pathways achieved the de novo production of these two compounds. Besides, this mutant has great potential for GA or pyrogallol derivatives production. The screening method could be used for other GA biosynthesis-related enzymes.

Recent Advancements in Lignin Valorization, Bioengineering, Catalysis and Biorefining

“…Overexpressing shikimate reductase from Juglans regia (walnut) from Ecoli causes a fivefold rise in gallate contents in tobacco plants, according to Chen, Chen, Yu, and Huo [3] on the biosynthesis of pyrogallol groups. In a recent study, we found that the Saccharothrix espanaensis bacteria coumarate 3-hydroxylase SAM5 could caffeate in order to produce 3, 4 , or 5-trihydroxycinnamate whenever produced in Ecoli.…”