Metabolic engineering of Streptomyces explomaris for increased production of the reverse antibiotic nybomycin

Abstract

Background Nybomycin is a reverse antibiotic with selective activity against fluoroquinolone-resistant Gram-positive bacteria, including Staphylococcus aureus, making it a promising candidate to fight against antimicrobial resistance. However, its clinical development has been limited by the low production yields of native producers. To address this, we explored the heterologous expression of the nybomycin biosynthetic gene cluster (nyb) from the natural producer S. albus subsp. chlorinus NRRL B-24,108 in different marine and terrestrial Streptomyces hosts, aiming to boost production through targeted regulatory and metabolic engineering. We also evaluated the use of seaweed-derived hydrolysates as sustainable fermentation substrates. Results Among several tested hosts, S. explomaris carrying the nyb gene cluster, produced the highest nybomycin titers. Global transcriptomic analysis identified transcriptional repression and precursor limitation as key bottlenecks. Deletion of the repressors nybW and nybX (NYB-1) significantly increased production, while further overexpression of genes boosting precursor supply (zwf2, nybF) led to the creation of NYB-3B, which reached a titer of 57 mg L−1 — fivefold higher than the previous benchmark. When cultivated on hydrolysates from commercial brown seaweed (Himanthalia elongata) without nutrient supplementation, NYB-3B achieved a titer of 14.8 mg L−1 . Conclusions This study demonstrates the potential of S. explomaris as a chassis for high-level heterologous nybomycin production and its compatibility with renewable marine feedstocks. Regulatory and metabolic engineering effectively relieved key bottlenecks and improved precursor supply. The use of seaweed hydrolysates supports the development of sustainable nybomycin production. Collectively, these findings provide a valuable foundation for future efforts toward improved supply and clinical development of nybomycin.