Combining Precursor-Directed Engineering with Modular Designing: An Effective Strategy for De Novo Biosynthesis of l‑DOPA in Bacillus licheniformis

3-Hydroxy-l-tyrosine (l-DOPA) is a promising drug for treating Parkinson’s disease. Tyrosine hydroxylase catalyzes the microbial synthesis of l-DOPA, which is hindered by the efficiency of catalysis, the supply of cofactor tetrahydrobiopterin, and the regulation of the pathway. In this study, the modular engineering strategy in Bacillus licheniformis was identified to effectively enhance l-DOPA production. First, the catalytic efficiency of biocatalyst tyrosine hydroxylase from Streptosporangium roseum DSM 43021 (SrTH) was improved by 20.3% by strengthening its affinity toward tetrahydrobiopterin. Second, the tetrahydrobiopterin supply pool was increased by bottleneck gene expression, oxygen transport facilitation, budC (encoding meso-2,3-butanediol dehydrogenase) deletion, and tetrahydrobiopterin regeneration using a native YfkO nitroreductase. The strain 45ABvC successfully produced tetrahydrobiopterin, which was detected as pterin (112.48 mg/L), the oxidation product of tetrahydrobiopterin. Finally, the yield of precursor l-tyrosine reached 148 mg/gDCW, with an increase of 71%, with the deletion of a novel spliced transcript 41sRNA associated with the regulation of the shikimate pathway. The engineered strain 45ABvCS::PD produced 167.14 mg/L (2.41 times of wild-type strain) and 1290 mg/L l-DOPA in a shake flask and a 15 L bioreactor, respectively, using a fermentation strategy on a mixture of carbon sources. This study holds great potential for constructing a microbial source of l-DOPA and its high-value downstream pharmaceuticals.