Inactivation of the sco2730/2731 copper chaperone–transporter system in Streptomyces coelicolor and its orthologs in Streptomyces venezuelae, together with chromosomal end deletion, greatly enhances secondary metabolism

Activating silent biosynthetic gene clusters (BGCs) in Streptomyces remains a major challenge in harnessing their vast secondary metabolic potential and requires diverse, complementary strategies, including bacterial co-cultivation, heterologous expression, chemical elicitation, modulation of gene expression, and the use of pleiotropic and pathway-specific genetic regulators, such as those influenced by cytosolic copper levels. Previous studies reported that, in S. coelicolor, disruption of the sco2730/2731 copper chaperone–transporter system (Sc-M1 mutant) markedly enhances secondary metabolism. However, this activation is only partially reproduced by antisense knockdown constructs targeting sco2730/sco2731 in S. coelicolor (Sc-M2 mutant) and other species (S. venezuelae, S. albidoflavus). This study investigates the basis of the strong activation observed in Sc-M1, with the aim of exploiting this mechanism for activating silent BGCs in Streptomyces. Genomic analysis revealed that, in addition to sco2730/2731 inactivation, the Sc-M1 mutant possesses a spontaneous deletion of both chromosomal ends. Construction of a sco2730 knockout mutant (Δsco2730, also affecting sco2731; Sc-M3 mutant) showed an effect on secondary metabolism comparable to that of the Sc-M2 mutant, and demonstrated that sco2730 disruption increases chromosomal-end instability. Metabolomic analyses showed that inactivation of sco2730/2731 (Sc-M3 mutant) or chromosomal-end deletion (Sc-M4 mutant) individually enhanced secondary metabolism. However, only the combination of Δsco2730 and chromosomal-end deletion (Sc-M5 mutant) approached the extensive metabolic activation observed in Sc-M1, affecting up to 60 secondary-metabolite adducts from 17 biosynthetic pathways. Similar synergistic effects were observed in S. venezuelae, where combined knockdown of the sco2730/2731 orthologues and chromosomal-end deletion strongly modulated secondary metabolism, repressing chloramphenicol production while inducing pikromycin biosynthesis, a typically silent and difficult-to-activate S. venezuelae BGC. Simultaneous disruption of the sco2730/31 copper chaperone–transporter system and chromosomal-end deletion synergistically enhance secondary metabolism production in S. coelicolor and S. venezuelae. This combined genetic manipulation provides a novel strategy for the challenging task of activating silent biosynthetic pathways and for potentially discovering new bioactive compounds across Streptomyces species.