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J Ind Microbiol Biotechnol. 2019 Sep 18. doi: 10.1007/s10295-019-02237-8. [Epub ahead of print]

Integrating enzyme evolution and high-throughput screening for efficient biosynthesis of L-DOPA.

Zeng W1,2, Xu B1,2,3, Du G1,4, Chen J1,2,3, Zhou J5,6,7.

Author information

1
Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
2
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
3
Jiangsu Provisional Research Center for Bioactive Product Processing Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
4
The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
5
Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China. zhoujw1982@jiangnan.edu.cn.
6
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China. zhoujw1982@jiangnan.edu.cn.
7
Jiangsu Provisional Research Center for Bioactive Product Processing Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China. zhoujw1982@jiangnan.edu.cn.

Abstract

L-DOPA is a key pharmaceutical agent for treating Parkinson's, and market demand has exploded due to the aging population. There are several challenges associated with the chemical synthesis of L-DOPA, including complicated operation, harsh conditions, and serious pollution. A biocatalysis route for L-DOPA production is promising, especially via a route catalyzed by tyrosine phenol lyase (TPL). In this study, using TPL derived from Erwinia herbicola (Eh-TPL), a mutant Eh-TPL was obtained by integrating enzyme evolution and high-throughput screening methods. L-DOPA production using recombinant Escherichia coli BL21 (DE3) cells harbouring mutant Eh-TPL was enhanced by 36.5% in shake flasks, and the temperature range and alkali resistance of the Eh-TPL mutant were promoted. Sequence analysis revealed two mutated amino acids in the mutant (S20C and N161S), which reduced the length of a hydrogen bond and generated new hydrogen bonds. Using a fed-batch mode for whole-cell catalysis in a 5 L bioreactor, the titre of L-DOPA reached 69.1 g L-1 with high productivity of 11.52 g L-1 h-1, demonstrating the great potential of Eh-TPL variants for industrial production of L-DOPA.

KEYWORDS:

Catalytic activity; Error-prone PCR; Fed-batch mode; High-throughput screening; Tyrosine phenol lyase

PMID:
31535250
DOI:
10.1007/s10295-019-02237-8

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