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

  • Wei Yuan
  • Shuang Zhong
  • Yanming Xiao
  • Zhao Wang
  • Jie SunEmail author
Article

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.

Keywords

Tyrosine phenol-lyase L-DOPA Kluyvera intermedia Random mutagenesis Structural analysis 

Notes

Funding Information

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).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflicts of interest.

Research Involving Human Participants and/or Animals

This study is not involving any human participants and/or animals.

Informed Consent

All the authors mentioned in the manuscript have agreed for authorship, read and approved the manuscript, and given consent for submission and subsequent publication of the manuscript.

Supplementary material

12010_2019_3164_MOESM1_ESM.pdf (0 kb)
Supplementary file S1: A multiple sequence alignment of homologous proteins colored according to residue conservation. (PDF 32 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and BioengineeringZhejiang University of TechnologyHangzhouChina
  2. 2.Biocatalysis and Transformation Engineering Research Center of Zhejiang ProvinceChangxing Pharmaceutical Co., LtdChangxingChina

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