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Nybomycin is an antibiotic compound with proven activity against multi-resistant Staphylococcus aureus , making it an interesting candidate for combating these globally threatening pathogens. For exploring its potential, sufficient amounts of nybomycin and its derivatives must be synthetized to fully study its effectiveness, safety profile, and clinical applications. As native isolates only accumulate low amounts of the compound, superior producers are needed. The heterologous cell factory S. albidoflavus 4N24, previously derived from the cluster-free chassis S. albidoflavus Del14, produced 860 μg L−1 of nybomycin, mainly in the stationary phase. A first round of strain development modulated expression of genes involved in supply of nybomycin precursors under control of the common P erm* promoter in 4N24, but without any effect. Subsequent studies with mCherry reporter strains revealed that P erm* failed to drive expression during the product synthesis phase but that use of two synthetic promoters (P kasOP* and P 41) enabled strong constitutive expression during the entire process. Using P kasOP* , several rounds of metabolic engineering successively streamlined expression of genes involved in the pentose phosphate pathway, the shikimic acid pathway, supply of CoA esters, and nybomycin biosynthesis and export, which more than doubled the nybomycin titer to 1.7 mg L−1 in the sixth-generation strain NYB-6B. In addition, we identified the minimal set of nyb genes needed to synthetize the molecule using single-gene-deletion strains. Subsequently, deletion of the regulator nybW enabled nybomycin production to begin during the growth phase, further boosting the titer and productivity. Based on RNA sequencing along the created strain genealogy, we discovered that the nyb gene cluster was unfavorably downregulated in all advanced producers. This inspired removal of a part and the entire set of the four regulatory genes at the 3′-end nyb of the cluster. The corresponding mutants NYB-8 and NYB-9 exhibited marked further improvement in production, and the deregulated cluster was combined with all beneficial targets from primary metabolism. The best strain, S. albidoflavus NYB-11, accumulated up to 12 mg L−1 nybomycin, fifteenfold more than the basic strain. The absence of native gene clusters in the host and use of a lean minimal medium contributed to a selective production process, providing an important next step toward further development of nybomycin. • Nybomycin is a promising antibiotic against multi-resistant S. aureus. • Genomic studies identified the minimal nybomycin gene cluster. • Streamlined chassis Streptomyces albidoflavus Del14 used for production. • Systematic engineering of primary and secondary pathways. • Engineered S. albidoflavus NYB-11 forms nybomycin up to 12 mg L−1 [ABSTRACT FROM AUTHOR]