Continued evolution of multidrug resistant pathogenic bacteria is sustaining a need for novel antibiotics with mechanisms distinct from those currently used. Streptomyces is a genus of filamentous soil dwelling bacteria with incredible genomic diversity and is a repertoire for biosynthesized antimicrobial compounds. Naturally, biosynthetic gene cluster (BGC) expression in Streptomyces is reserved for a secondary metabolic phase where antimicrobial compounds are synthesized as a biological warfare strategy during nutrient competition. Identifying novel BGCs and characterising their product is often constrained by the initial challenge of eliciting their expression. Here, we undertook a detail temporal proteome characterisation of Streptomyces hygroscopicus NRRL 30439 during submerged culture fermentation with the aim of elucidating the capabilities of its secondary metabolome.
PacBio RSII sequencing of S. hygroscopicus NRRL 30439 allowed us to construct a single 9.7 Mb linear genome and identify 24 biosynthetic regions using antiSMASH v5.1.0. A chemically defined media was developed to elicit a diverse nutrient stress response, which was captured by temporal intracellular proteomics. Data was acquired using a Thermo Fisher Q Exactive HF-X in DIA mode, and analysed with Spectronaut. Of the 24 predicted BGCs, 6 were found to express all their core BGC proteins where most of these proteins exhibited expression profiles coincident with nutrient depletion. Well-studied regulators of morphological differentiation (bld cascade) were detected in the proteome, suggesting that S. hygroscopicus NRRL 30439 regulates facets of secondary metabolism by selective expression of tRNAleuUUA. This is further supported in the proteome and exemplified in cases where TTA codon-containing BGC genes exhibiting stress-induced expression, whilst adjacent genes of the same cluster exhibit an earlier onset of expression. While many of the product molecules of predicted BGCs remain unknown, our work represents a method of eliciting secondary metabolism in Streptomyces and identification of likely-expressed novel candidate molecules. This allows for a narrowed focus when proceeding to characterize novel antimicrobial candidates.