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

Area of Science:

• Biocatalysis and Enzyme Engineering
• Pharmaceutical Chemistry
• Metabolic Engineering

Background:

• L-DOPA is essential for Parkinson's disease treatment, with rising global demand.
• Chemical synthesis of L-DOPA faces challenges like complexity, harsh conditions, and environmental pollution.
• Biocatalysis using tyrosine phenol lyase (TPL) presents a promising alternative for L-DOPA production.

Purpose of the Study:

• To engineer a more robust and efficient tyrosine phenol lyase (TPL) from Erwinia herbicola (Eh-TPL) for enhanced L-DOPA production.
• To improve the enzyme's operational stability, including its temperature range and alkali resistance.
• To demonstrate the industrial feasibility of the engineered enzyme for large-scale L-DOPA synthesis.

Main Methods:

• Enzyme evolution and high-throughput screening were employed to obtain a mutant Eh-TPL.
• Recombinant Escherichia coli BL21 (DE3) cells expressing the mutant Eh-TPL were used for whole-cell biocatalysis.
• Fed-batch fermentation in a 5 L bioreactor was optimized for L-DOPA production.

Main Results:

• The engineered Eh-TPL mutant enhanced L-DOPA production by 36.5% in shake flasks compared to the wild type.
• The mutant exhibited improved temperature and alkali resistance.
• A high L-DOPA titre of 69.1 g/L and productivity of 11.52 g/L/h were achieved in a 5 L bioreactor using fed-batch whole-cell catalysis.
• Sequence analysis identified S20C and N161S mutations affecting hydrogen bonding within the enzyme.

Conclusions:

• The engineered Eh-TPL variant demonstrates significantly improved performance for L-DOPA production.
• The enhanced enzyme stability and high yields highlight its potential for industrial-scale, sustainable L-DOPA manufacturing.
• This biocatalytic approach offers a viable and environmentally friendly alternative to conventional chemical synthesis methods.