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Appl Microbiol Biotechnol. 2018 Mar;102(5):2129-2141. doi: 10.1007/s00253-018-8741-y. Epub 2018 Jan 19.

Efficient biosynthesis of L-phenylglycine by an engineered Escherichia coli with a tunable multi-enzyme-coordinate expression system.

Author information

1
The Key Laboratory of Industrial Biotechnology, Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, 214122, China.
2
The Key Laboratory of Industrial Biotechnology, Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, 214122, China. raozhm@jiangnan.edu.cn.

Abstract

Whole-cell catalysis with co-expression of two or more enzymes in a single host as a simple low-cost biosynthesis method has been widely studied and applied but hardly with regulation of multi-enzyme expression. Here we developed an efficient whole-cell catalyst for biosynthesis of L-phenylglycine (L-Phg) from benzoylformic acid through co-expression of leucine dehydrogenase from Bacillus cereus (BcLeuDH) and NAD+-dependent mutant formate dehydrogenase from Candida boidinii (CbFDHA10C) in Escherichia coli with tunable multi-enzyme-coordinate expression system. By co-expressing one to four copies of CbFDHA10C and optimization of the RBS sequence of BcLeuDH in the expression system, the ratio of BcLeuDH to CbFDH in E. coli BL21/pETDuet-rbs 4 leudh-3fdh A10C was finally regulated to 2:1, which was the optimal one determined by enzyme-catalyzed synthesis. The catalyst activity of E. coli BL21/pETDuet-rbs 4 leudh-3fdh A10C was 28.4 mg L-1 min-1 g-1 dry cell weight for L-Phg production using whole-cell transformation, it's was 3.7 times higher than that of engineered E. coli without enzyme expression regulation. Under optimum conditions (pH 8.0 and 35 °C), 60 g L-1 benzoylformic acid was completely converted to pure chiral L-Phg in 4.5 h with 10 g L-1 dry cells and 50.4 g L-1 ammonium formate, and with enantiomeric excess > 99.9%. This multi-enzyme-coordinate expression system strategy significantly improved L-Phg productivity and demonstrated a novel low-cost method for enantiopure L-Phg production.

KEYWORDS:

Formate dehydrogenase; L-Phenylglycine; Leucine dehydrogenase; Tunable multi-enzyme-coordinate expression; Whole-cell catalysis

PMID:
29352398
DOI:
10.1007/s00253-018-8741-y
[Indexed for MEDLINE]

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