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Background <p>Activating silent biosynthetic gene clusters (BGCs) in <i>Streptomyces</i> 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 <i>S. coelicolor</i>, disruption of the <i>sco2730/2731</i> copper chaperone–transporter system (Sc-M1 mutant) markedly enhances secondary metabolism. However, this activation is only partially reproduced by antisense knockdown constructs targeting <i>sco2730/sco2731</i> in <i>S. coelicolor</i> (Sc-M2 mutant) and other species (<i>S. venezuelae</i>, <i>S. albidoflavus</i>). 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 <i>Streptomyces</i>.</p> Results <p>Genomic analysis revealed that, in addition to <i>sco2730/2731</i> inactivation, the Sc-M1 mutant possesses a spontaneous deletion of both chromosomal ends. Construction of a <i>sco2730</i> knockout mutant (Δ<i>sco2730</i>, also affecting <i>sco2731</i>; Sc-M3 mutant) showed an effect on secondary metabolism comparable to that of the Sc-M2 mutant, and demonstrated that <i>sco2730</i> disruption increases chromosomal-end instability. Metabolomic analyses showed that inactivation of <i>sco2730/2731</i> (Sc-M3 mutant) or chromosomal-end deletion (Sc-M4 mutant) individually enhanced secondary metabolism. However, only the combination of Δ<i>sco2730</i> 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 <i>S. venezuelae</i>, where combined knockdown of the <i>sco2730/2731</i> orthologues and chromosomal-end deletion strongly modulated secondary metabolism, repressing chloramphenicol production while inducing pikromycin biosynthesis, a typically silent and difficult-to-activate <i>S. venezuelae</i> BGC.</p> Conclusions <p>Simultaneous disruption of the <i>sco2730/31</i> copper chaperone–transporter system and chromosomal-end deletion synergistically enhance secondary metabolism production in <i>S. coelicolor</i> and <i>S. venezuelae</i>. 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 <i>Streptomyces</i> species.</p>

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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

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  • 发表日期：

    2026年4月6日

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  • Gemma Fernández-García,
  • Paula Valdés-Chiara,
  • Paula García-Cancela,
  • Nathaly González-Quiñónez,
  • Juan Serna-Diestro,
  • Felipe Lombó,
  • María Montes-Bayón,
  • Ángel Manteca

摘要

Background

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.

Results

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.

Conclusions

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.