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Efficient biocatalyst of L-DOPA with Escherichia coli expressing a tyrosine phenol-lyase mutant from Kluyvera intermedia

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Abstract

L-DOPA (L-dihydroxyphenylalanine) is a promising drug for Parkinson’s disease and thereby has a growing annual demand. Tyrosine phenol-lyase (TPL)–based catalysis is considered to be a low-cost yet efficient route for biosynthesis of L-DOPA. TPL is a tetrameric enzyme that catalyzes the synthesis of L-DOPA from pyrocatechol, sodium pyruvate, and ammonium acetate. The implementation of TPL for L-DOPA production has been hampered and the need for the most efficient TPL source with higher L-DOPA production and substrate conversion rate is prevailing. This study involves identifying a novel TPL from Kluyvera intermedia (Ki-TPL) and displayed a robust expression in Escherichia coli. The recombinant strain YW000 carrying Ki-TPL proved strong catalytic activity with a highest L-DOPA yield compared with 16 other TPLs from different organisms. With a further aim to improve this efficiency, random mutagenesis of Ki-TPL was performed and a mutant namely YW021 was obtained. The whole cells of YW021 as biocatalyst yielded 150.4 g L−1 of L-DOPA with a 99.99 % of pyrocatechol conversion at the optimum condition of pH 8.0 at 25 °C, which is the highest level reported to date. Further, the homology modeling and structural analysis revealed the mutant residues responsible for the extensive L-DOPA biosynthesis.

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Funding

This study was financially supported by the Natural Science Foundation of Zhejiang Province (Grant No. LQ18C010006 and LY19C010005) and National Natural Science Foundation of China (Grant Number: 31900497).

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Authors and Affiliations

  1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China

    Wei Yuan, Shuang Zhong, Zhao Wang & Jie Sun

  2. Biocatalysis and Transformation Engineering Research Center of Zhejiang Province, Changxing Pharmaceutical Co., Ltd, Changxing, 313100, China

    Yanming Xiao

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  1. Wei Yuan
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  3. Yanming Xiao
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  4. Zhao Wang
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Correspondence to Jie Sun.

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Supplementary file S1:

A multiple sequence alignment of homologous proteins colored according to residue conservation. (PDF 32 kb)

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Yuan, W., Zhong, S., Xiao, Y. et al. Efficient biocatalyst of L-DOPA with Escherichia coli expressing a tyrosine phenol-lyase mutant from Kluyvera intermedia. Appl Biochem Biotechnol 190, 1187–1200 (2020). https://doi.org/10.1007/s12010-019-03164-1

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