Multilevel metabolic engineering of Bacillus amyloliquefaciens for enhanced L-tyrosine production

L-Tyrosine is a valuable aromatic amino acid with diverse applications. Although plasmid-based overexpression has markedly improved L-tyrosine production, the dependence on antibiotic selection and genetic instability under nonselective conditions limits its industrial applicability. To address these issues, this study employed a multi-level metabolic engineering strategy to enhance the L-tyrosine biosynthetic pathway at the genomic level in Bacillus amyloliquefaciens. First, an expression cassette containing the mutant prephenate dehydrogenase (TyrA^I309A/E330V) was inserted into the genome of the wild-type strain Z1. When the copy number reached three (strain Z4), L-tyrosine production reached 437.43mg/L. Subsequently, the mutant DAHP synthase (AroA^R27A/K38A) was integrated, and the resulting two-copy strain Z7 increased L-tyrosine production to 591.29mg/L. Systematic identification of rate-limiting steps showed that most pathway genes enhanced production to varying extents. A promoter in situ replacement strategy was then applied to construct strain Z21, which further increased the titer to 813.01mg/L. Finally, by optimizing precursor supply, the engineered strain Z32 reached an L-tyrosine titer of 1079.94mg/L. Overall, this plasmid-free and antibiotic-independent platform offers a promising strategy for stable biosynthesis of L-tyrosine and other aromatic amino acids in microbial cell factories.