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Natural constitutive promoters from Actinoplanes siamensis TBRC 4237 enable high-level heterologous gene expression in Streptomyces coelicolor

ABSTRACT: Activation of cryptic biosynthetic gene clusters (BGCs) in actinomycetes often requires strong and reliable promoters, yet native promoters from rare actinomycetes remain scarce. Here, we used transcriptome analysis of Actinoplanes siamensis TBRC 4237 to identify highly expressed genes and selected 22 upstream regions as candidate constitutive promoters. Their activities were evaluated in Streptomyces coelicolor M1146 using indigoidine synthetase and latex-clearing protein (LCP) as heterologous reporters. Thirteen promoters drove indigoidine production at levels exceeding those of kasOp* and ermEp*, the widely used strong constitutive promoters in Streptomyces. Several promoters also yielded robust LCP activity, with promoter 650A achieving the highest expression across both reporter systems. Motif analysis showed no clear correlation between predicted -10/-35 elements and promoter strength, indicating additional regulatory features. These natural promoters provide valuable genetic parts for heterologous expression, BGC activation, and pathway refactoring in actinomycetes.

ORGANISM(S): Actinoplanes siamensis

PROVIDER: GSE320076 | GEO | 2026/02/25

REPOSITORIES: GEO

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Project description:Biosynthetic gene clusters (BGCs) encoding the production of bacteriocins are widespread among bacterial isolates and are important genetic determinants of competitive fitness within a given habitat. Staphylococci produce a tremendous diversity of compounds, and the corresponding BGCs are frequently associated with mobile genetic elements, suggesting gain and loss of biosynthetic capacity. Pharmaceutical biology has shown that compound production in heterologous hosts is often challenging, and many BGC recipients initially produce small amounts of compound or show reduced growth rates. To assess whether transfer of BGCs between closely related Staphylococcus aureus strains can be instantly effective or requires elaborate metabolic adaptation, we investigated the intraspecies transfer of a BGC encoding the ribosomally synthesized and posttranslationally modified peptide (RiPP) micrococcin P1 (MP1). We found that acquisition of the BGC by S. aureus RN4220 enabled immediate MP1 production but also imposed a metabolic burden, which was relieved after prolonged cultivation by adaptive mutation. We used a multiomics approach to study this phenomenon and found adaptive evolution to select for strains with increased activity of the tricarboxylic acid cycle (TCA), which enhanced metabolic fitness and levels of compound production. Metabolome analysis revealed increases of central metabolites, including citrate and α-ketoglutarate in the adapted strain, suggesting metabolic adaptation to overcome the BGC-associated growth defects. Our results indicate that BGC acquisition requires genetic and metabolic predispositions, allowing the integration of bacteriocin production into the cellular metabolism. Inappropriate metabolic characteristics of recipients can entail physiological burdens, negatively impacting the competitive fitness of recipients within natural bacterial communities.IMPORTANCE: Human microbiomes are critically associated with human health and disease. Importantly, pathogenic bacteria can hide in human-associated communities and can cause disease when the composition of the community becomes unbalanced. Bacteriocin-producing commensals are able to displace pathogens from microbial communities, suggesting that their targeted introduction into human microbiomes might prevent pathogen colonization and infection. However, to develop probiotic approaches, strains are needed that produce high levels of bioactive compounds and retain cellular fitness within mixed bacterial communities. Our work offers insights into the metabolic burdens associated with the production of the bacteriocin micrococcin P1 and highlights evolutionary strategies that increase cellular fitness in the context of production. Metabolic adaptations are most likely broadly relevant for bacteriocin producers and need to be considered for the future development of effective microbiome editing strategies.

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Natural constitutive promoters from Actinoplanes siamensis TBRC 4237 enable high-level heterologous gene expression in Streptomyces coelicolor

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