The search for novel bioactive compounds to overcome resistance to current therapeutics has become of utmost importance.Streptomycesspp. are one of the main sources of bioactive compounds currently used in medicine. In this work, five different global transcriptional regulators and five housekeeping genes, known to induce the activation or overproduction of secondary metabolites inStreptomyces coelicolor, were cloned in two separated constructs and expressed in 12 different strains ofStreptomycesspp. from the in-house CS collection. These recombinant plasmids were also inserted into streptomycin and rifampicin resistantStreptomycesstrains (mutations known to enhance secondary metabolism inStreptomyces). Different media with diverse carbon and nitrogen sources were selected to assess the strains’ metabolite production. Cultures were then extracted with different organic solvents and analysed to search for changes in their production profiles. An overproduction of metabolites already known to be produced by the biosynthesis wild-type strains was observed such as germicidin by CS113, collismycins by CS149 and CS014, or colibrimycins by CS147. Additionally, the activation of some compounds such as alteramides in CS090a pSETxkBMRRH and CS065a pSETxkDCABA or inhibition of the biosynthesis of chromomycins in CS065a in pSETxkDCABA when grown in SM10 was demonstrated. Therefore, these genetic constructs are a relatively simple tool to manipulateStreptomycesmetabolism and explore their wide secondary metabolites production potential.