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

5 years ago

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

Qipeng Yuan, Xiaolin Shen, Yajun Yan, Jia Wang, Jian Wang, Zhenya Chen

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;9999: 1–10. © 2017 Wiley Periodicals, Inc. A highly active PobA variant for hydroxylating 3,4-DHBA into GA via structure-based protein engineering approach was proposed. Both in vitro enzyme assays and in vivo conversion experiments were carried out to examine this hypothesis and the results confirmed the rational analysis. Subsequently, this new mutant was employed to achieve efficient GA de novo production.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/bit.26364