期刊
BIOTECHNOLOGY AND BIOENGINEERING
卷 114, 期 11, 页码 2571-2580出版社
WILEY
DOI: 10.1002/bit.26364
关键词
gallic acid; aromatic compounds; shikimate pathway; p-hydroxybenzoate hydroxylase; protein engineering; microbial synthesis
资金
- National Natural Science Foundation of China [21406010, 21606012, 21636001]
- Introducing Talents of Discipline to Universities
- 111'' project [B13005]
- Changjiang Scholars and Innovative Research Team in Universities in China [IRT13045]
- Academic Leader of Beijing Polytechnic [DTR201601]
- Key Project of Beijing Polytechnic [YZK028]
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 1149.59mg/L GA from 1000mg/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 440.53mg/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 1266.39mg/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. (C) 2017 Wiley Periodicals, Inc.
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