The design and engineering of secondary metabolite gene clusters that are characterized by complicated genetic organization, require the development of collections of well-characterized genetic control elements that can be reused reliably. Although a few intrinsic terminators and RBSs are used routinely, their translation and termination efficiencies have not been systematically studied in Actinobacteria. Here, we analyzed the influence of the regions surrounding RBSs on gene expression in these bacteria. We demonstrated that inappropriate RBSs can reduce the expression efficiency of a gene to zero. We developed a genetic device – an in vivo RBS-selector – that allows selection of an optimal RBS for any gene of interest, enabling rational control of the protein expression level. In addition, a genetic tool that provides the opportunity for measurement of termination efficiency was developed. Using this tool, we found strong terminators that lead to a 17–100-fold reduction in downstream expression and are characterized by sufficient sequence diversity to reduce homologous recombination when used with other elements. For the first time, a C-terminal degradation tag was employed for the control of protein stability in Streptomyces. Finally, we describe a collection of regulatory elements that can be used to control metabolic pathways in Actinobacteria.

We report here a transposon-based strategy to generate Streptomyces globisporus 1912 mutants with improved landomycin E production. The modified minitransposon with strong, outward-oriented promoters for the overexpression of downstream-situated genes has been applied for mutant library generation. Approximately 2500 mutants of S. globisporus 1912 were analyzed for landomycin E production, leading to the identification of several overproducers. Subcloning and sequencing of the sites of integration showed that some of the inactivated genes encode proteins with a similarity to known bacterial regulators such as TetR and LuxR families. One of the regulators (GntR type) has shown the strongest influence on the landomycin E production. Its ortholog (encoded by sco3269) in Streptomyces coelicolor was characterized in greater detail and showed similar effects on actinorhodin production and morphological differentiation.

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