Genome-Scale Modeling–Guided Metabolic Engineering Enables Heterologous Production of 3,4-Amino-4-Hydroxybenzoic Acid in <em>Streptomyces thermoviolaceus</em>

3-Amino-4-hydroxybenzoic acid (3,4-AHBA) is a non-proteinogenic aromatic compound that functions as a key biosynthetic precursor for diverse secondary metabolites with pharmaceutical and industrial value. Microbial production of 3,4-AHBA offers a sustain-able alternative to petroleum-based chemical synthesis; however, metabolic complexity and trade-offs between growth and product formation constrain rational strain design. Here, genome-scale metabolic (GSM) modeling and flux balance analysis (FBA) were in-tegrated with targeted genetic engineering to elucidate and enhance 3,4-AHBA production in Streptomyces thermoviolaceus. A genome-scale metabolic model was constructed and ex-panded by incorporating the nspH–nspI gene operon, which encodes the 3,4-AHBA bio-synthetic pathway. In silico FBA predicted substantial rewiring of central carbon metabo-lism, with carbon flux redirected from glycolysis and the tricarboxylic acid cycle toward aspartate-derived intermediates and 3,4-AHBA synthesis, accompanied by reduced bio-mass-associated flux. Guided by these predictions, an engineered strain (St::NspHI) was developed and experimentally evaluated. Consistent with model predictions, the engi-neered strain exhibited lower growth rates and glucose uptake than the wild type, reflect-ing a metabolic burden. Nevertheless, 3,4-AHBA production was achieved exclusively in the engineered strain. Comparison of simulated and experimental fluxes revealed overes-timation by FBA, likely due to secondary metabolism and incomplete genome annotation. Overall, GSM-guided design enables optimization of precursor production.