Enhanced Natamycin production in Streptomyces gilvosporeus through phosphate tolerance screening and transcriptome-based analysis of high-yielding mechanisms

Natamycin is a natural antibiotic with broad-spectrum antifungal activity, widely used in food preservation, medicine, and biological control. However, the relatively low biosynthetic capacity of producing strains limits further industrialization and broader applications of natamycin. Due to the complexity of cellular metabolism, evolutionary engineering is required for developing strains with enhanced natamycin biosynthetic capacity. Here, protoplast fusion combined with phosphate tolerance screening was employed for the first time to enhance natamycin production of Streptomyces gilvosporeus. A high-yielding strain, GR-2, was obtained, with natamycin production twice that of the original strain. Transcriptomic analysis revealed that the natamycin biosynthetic gene cluster and several primary metabolic pathways were significantly upregulated in GR-2, likely contributing to its high production performance. Further experiments, including amino acid addition and reverse engineering, confirmed that branched-chain amino acid, nitrogen, and phosphate metabolism play crucial roles in promoting natamycin production. Silencing of the phosphate metabolism transcriptional regulators PhoP and PhoR led to a decreased expression of natamycin biosynthetic genes and significantly reduced natamycin production, highlighting the key role of these regulators in S. gilvosporeus. Based on omics data, co-expression of phoP and phoR in GR-2 resulted in the engineered strain GR2-P3, which exhibited a 25% increase in natamycin production in shake flasks. In a 5 L fermenter, GR2-P3 achieved a natamycin production of 12.2 ± 0.6 g·L⁻¹, the highest yield reported for S. gilvosporeus to date. Our findings suggest that the high production performance of GR-2 is primarily due to the upregulation of the natamycin biosynthetic gene cluster and genes related to precursor supply. Increasing the intracellular supply of valine and glutamate significantly enhanced natamycin production. Additionally, the natamycin biosynthetic gene cluster is likely positively regulated by PhoP and PhoR. Our work presents a novel strategy for strain screening and evolution to improve natamycin production and identifies novel molecular targets for metabolic engineering.