Rational engineering of p‐hydroxybenzoate hydroxylase to enable efficient gallic acid synthesis via a novel artificial biosynthetic pathway

Abstract

Gallic acid (GA) is a naturally occurring phytochemical that has strong antioxidant and antibacterial activities. It is also used as a potential platform chemical for the synthesis of diverse high-value compounds. Hydrolytic degradation of tannins by acids, bases or microorganisms serves as a major way for GA production, which however, might cause environmental pollution and low yield and efficiency. Here, we report a novel approach for efficient microbial production of GA. First, structure-based rational engineering of PobA, a p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa, generated a new mutant, Y385F/T294A PobA, which displayed much higher activity toward 3,4-dihydroxybenzoic acid (3,4-DHBA) than the wild-type and any other reported mutants. Remarkably, expression of this mutant in Escherichia coli enabled generation of 1149.59 mg/L GA from 1000 mg/L 4-hydroxybenzoic acid (4-HBA), representing a 93% molar conversion ratio. Based on that, we designed and reconstituted a novel artificial biosynthetic pathway of GA and achieved 440.53 mg/L GA production from simple carbon sources in E. coli. Further enhancement of precursor supply through reinforcing shikimate pathway was able to improve GA de novo production to 1266.39 mg/L in shake flasks. Overall, this study not only led to the development of a highly active PobA variant for hydroxylating 3,4-DHBA into GA via structure-based protein engineering approach, but also demonstrated a promising pathway for bio-based manufacturing of GA and its derived compounds. Biotechnol. Bioeng. 2017;114: 2571-2580. © 2017 Wiley Periodicals, Inc.

Expanding the repertoire of aromatic chemicals by microbial production

“…24,27 Recently, new reports have confirmed that gallic acid could be produced efficiently from protocatechuic acid by the introduction of the p-hydroxybenzoate hydroxylase (PobA) mutant (Fig. 28,29 The production of vanillin or its derivatives from renewable feedstocks has been achieved in several reports, but the cytotoxicity limits the yield of microbial production. 28,29 The production of vanillin or its derivatives from renewable feedstocks has been achieved in several reports, but the cytotoxicity limits the yield of microbial production.…”

“…However, the strain harboring target plasmids is genetically unstable compared with genome engineered strains and plasmid maintenance also is a metabolic burden toward the host cell, especially for high-copy number plasmids. 29 Flavin-dependent halogenase has strong substrate specificity and regioselectivity. The expression level of the target gene is determined by the strength of its promoter.…”

“…2). 28,29 The production of vanillin or its derivatives from renewable feedstocks has been achieved in several reports, but the cytotoxicity limits the yield of microbial production. 13,14 To overcome the toxicity of vanillin, a gene encoding family 1 UDP-glycosyltransferase from Arabidopsis thaliana was expressed in Schizosaccharomyces pombe and the engineered enzyme catalyzed the conversion of vanillin to vanillin -D-glucoside, which is not toxic to the yeast cells and thus may be accumulated in larger amounts.…”

“…Remarkably, expression of this mutant in E. coli enabled generation of 1149.59 mg L −1 gallic acid from 1000 mg L −1 4-hydroxybenzoic acid, representing a 93% molar conversion ratio. 29 Flavin-dependent halogenase has strong substrate specificity and regioselectivity. Using a structure-guided mutagenesis approach, the regioselectivity as well as the catalytic activity of PrnA were altered and the double mutant, PrnA E450K,F454k , was found to display a 16-fold increase in activity compared to the WT enzyme.…”

Homology models of mouse and rat estrogen receptor-α ligand-binding domain created by in silico mutagenesis of a human template: Molecular docking with 17β-estradiol, diethylstilbestrol, and paraben analogs

“…Moreover, the general notion of 4OH as a ligand in complex with proteins should not be surprising, given that it has been docked into human cyclooxygenase-2 (COX-2) [115], despite negative results in a COX-2 dependent human smooth muscle cell assay [116]. Furthermore, 4OH is found as a bound ligand in a variety of enzymes, including carbonic anhydrase [117], p -hydroxybenzoate hydroxylase [118], and 4-hydroxybenzoate octaprenyltransferase [119]. Lastly, 4OH was found to have intermediate antagonistic activity among paraben analogs against the human androgen receptor [25], and when its reported docking score was converted to an LE dock value, its relative rank increased from 7 th to 1 st place out of nine compounds, similar to the results we obtained in the present study.…”