Cost-Effective Production of L-DOPA by Tyrosinase-Immobilized Polyhydroxyalkanoate Nanogranules in Engineered Halomonas bluephagenesis TD01.

Jiping Zhao,Dan Tan,Ganqiao Ran,Mengmeng Xu,Xiaoyun Lu

doi:10.3390/molecules26133778

Abstract

3,4-dihydroxyphenyl-L-alanine (L-DOPA) is a preferred drug for Parkinson’s disease, with an increasing demand worldwide that mainly relies on costly and environmentally problematic chemical synthesis. Yet, biological L-DOPA production is unfeasible at the industrial scale due to its low L-DOPA yield and high production cost. In this study, low-cost Halomonas bluephagenesis TD01 was engineered to produce tyrosinase TyrVs-immobilized polyhydroxyalkanoate (PHA) nanogranules in vivo, with the improved PHA content and increased immobilization efficiency of TyrVs accounting for 6.85% on the surface of PHA. A higher L-DOPA-forming monophenolase activity of 518.87 U/g PHA granules and an L-DOPA concentration of 974.36 mg/L in 3 h catalysis were achieved, compared to those of E. coli. Together with the result of L-DOPA production directly by cell lysates containing PHA-TyrVs nanogranules, our study demonstrated the robust and cost-effective production of L-DOPA by H. bluephagenesis, further contributing to its low-cost industrial production based on next-generation industrial biotechnology (NGIB).

Highlights

More than 10 million people worldwide are living with Parkinson’s disease (PD), a prevalent degenerative disorder of the central nervous system that affects the nerve cells in the brain with reduced dopamine levels. 3,4-dihydroxyphenyl-L-alanine (L-DOPA) is currently the most promising drug for PD treatment [1] since it can cross the blood–brain barrier and functions as the precursor of dopamine, alleviating Parkinson’s disease [2]
Substantial efforts have been made to synthetize L-DOPA biologically using a variety of biocatalysts such as tyrosine phenollyase (Tpl) [7], tyrosinase (Tyr) [8], or p-hydroxyphenylacetate 3-hydroxylase (PHAH) [7,9], either by microbial fermentation and biotransformation in vivo or biocatalytic conversion in vitro
This study focuses on the engineering of the industrial host H. bluephagenesis TD01, aiming to promote high-yield and low-cost production of L-DOPA

Summary

Introduction

More than 10 million people worldwide are living with Parkinson’s disease (PD), a prevalent degenerative disorder of the central nervous system that affects the nerve cells in the brain with reduced dopamine levels. 3,4-dihydroxyphenyl-L-alanine (L-DOPA) is currently the most promising drug for PD treatment [1] since it can cross the blood–brain barrier and functions as the precursor of dopamine, alleviating Parkinson’s disease [2]. Regardless of whether the TyrVs-PhaCHb fusion protein was expressed in the plasmid (H.b-P-Vs) or genome (H.b-G-Vs), the cell growth and/or PHA content were greatly improved, compared to those of E. coli, and more than 20-fold increased concentration of PHA was achieved in H. bluephagenesis (Table 1), revealing the advantages of H. bluephagenesis as a good host for the synthesis of PHA nanogranules. TyrVs immobilized on the surface of PHA by integrated expression in the genome of H.b-G-Vs accounted for about 3.48% (Table S2 and Figure S4), which is still higher than that of E. coli but slightly lower than that of H.b-P-Vs strain, as the TyrVs-PhaCHb fusion protein was expressed in a higher-copy-number plasmid in H.b-P-Vs. The PHA-TyrVs nanogranules produced by H. bluephagenesis exhibited significantly increased content and immobilized TyrVs, which would benefit its activity and further synthesis of L-DOPA.

Discussions

Materials and Methods

Production and Purification of PHA-TyrVs Nanogranules

Findings

Conclusions