Abstract
The global incidence of Parkinson's disease continues to rise. Levodopa (l-DOPA) is the core therapeutic drug, and efficient and sustainable production methods are needed. However, the complex metabolic pathways and the low catalytic efficiency of enzymes limit biosynthesis of l-DOPA in microorganisms. To address this issue, this study significantly enhanced the production efficiency of l-DOPA through a multi-dimensional, integrated metabolic and enzyme engineering approach. Firstly, the de novo synthesis pathway for l-DOPA was established through optimization of the promoter, ribosome-binding site (RBS), plasmid copy number, and tighly accurately regulating the expression level of key enzymes. Secondly, combined with metabonomic analysis, carbon metabolic flow was diverted, increasing the l-DOPA titer by 36.7 %. Glucose dehydrogenase (BmgdH) and gluconate kinase (gntK) were introduced to construct a cofactor regeneration system, which synergistically enhanced the supply of NADH and FADH(2), increasing the l-DOPA conversion rate by 18 %. Next, the substrate tunnel of 4-hydroxyphenylacetic acid-3-monooxygenase subunit B (HpaB) was subjected to rational design, and mutant T292A significantly expanded the substrate channel, improved catalytic efficiency, and decreased l-tyrosine by 87 %. Finally, through the process optimization in a 5 L bioreactor (involving phased pH control and induction timing adjustment) achieved an l-DOPA titer of 60.73 g/L, the highest reported to date for de novo microbial synthesis. This research offers a novel approach for industrial biosynthesis of l-DOPA, and broadens engineering concepts for efficient synthesis of aromatic compounds.