A set of synthetic versatile genetic control elements for the efficient expression of genes in Actinobacteria

Abstract

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.

sRNA scr5239 Involved in Feedback Loop Regulation of Streptomyces coelicolor Central Metabolism

“…The western blot analysis shown in Figure 2A effectively confirms the TMT data with a two-fold increase in PEPCK expression in FE02 ( scr5239) and approximately two-fold decrease in FE03 (scr5239+) compared to the wild type (FE01). To exclude an effect on the transcription of PEPCK, we replaced the wild type pepck promoter in pPEPCK_F3 with the synthetic promoter SP4 (Horbal et al, 2018), thus generating the integrating plasmid pSP4_PEPCK_F3. Integration resulted in the respective strains FE04, FE05, and FE06 for M145, scr5239, and scr5239+, respectively.…”

Characterization and overproduction of cell-associated cholesterol oxidase ChoD from Streptomyces lavendulae YAKB-15

“…To avoid promoter leakage due to the read-through from the upstream genes (i.e. bacteriophage phi31 integrase and apramycin resistance genes) two strong terminator, a synthetic T4 kurz 52 and a natural terminator ECK120029600 53 were placed upstream of the promoter.…”

An Update on Molecular Tools for Genetic Engineering of Actinomycetes—The Source of Important Antibiotics and Other Valuable Compounds

“…These may have the potential to be applied in engineering of a wide range of actinomycetes. In addition, the already established systems (e.g., Streptomyces lividans strains containing glucuronidase (GusA)) were utilized for diverse modifications (e.g., variation of the distance between the Shine-Dalgarno (SD) domain and the start codon [ 134 ]) resulting in refactored as well as new synthetic promoters [ 135 , 136 , 137 , 138 , 139 ]. Their features and examples of successful applications for engineering of actinomycetes genomes were presented in several publications [ 72 , 74 , 129 , 131 , 134 , 140 , 141 , 142 , 143 ].…”