actinorhodin and indigoidine

Biosynthesis of secondary metabolites is a highly complex process that often requires tight control of their production, as overproduction of metabolites could be toxic and also may cause metabolic burden to their hosts. Tight control of metabolite production could be achieved by expressing key biosynthetic genes under control of an inducible regulatory system. In this study, we employed the modular design approach to build a high performance synthetic inducible regulatory system that displays a large dynamic range and thus is well-suited for the modulation of secondary metabolite production in Streptomyces. To this end, an inducible regulatory system was divided into three separate functional modules: (1) the induction module, (2) the target expression module, and (3) the repressor expression module. Then, these three separate modules were individually optimized in a stepwise manner and assembled to a new system. First, the cumate (CMT) induction module was chosen as the best performing induction module based on the large dynamic range and moderate inducer sensitivity. Then the CMT induction module maintained its performance when combined with diverse constitutive target expression modules, in which overall dynamic ranges varied depending on maximum promoter strengths. Lastly, the repressor expression module was optimized to achieve complete elimination of leaky expression, further increasing the dynamic range of the system. We also demonstrate that any strong constitutive regulatory system could be converted into an inducible regulatory system by simple CRISPR/Cas9-aided markerless insertion of an operator sequence whenever tight control of gene expression is required. We believe that the synthetic inducible regulatory system we report here would become a useful tool in modulating secondary metabolite production in Streptomyces.

Topics: Anthraquinones; CRISPR-Cas Systems; Escherichia coli; Gene Expression Regulation, Bacterial; Genetic Engineering; Multigene Family; Piperidones; Promoter Regions, Genetic; Saccharomyces cerevisiae; Secondary Metabolism; Streptomyces; Synthetic Biology; Transcription, Genetic

We previously developed an efficient deletion system for streptomycetes based on the positive selection of double-crossover events using bpsA, a gene for producing the blue pigment indigoidine. Using this system, we removed interfering secondary metabolite clusters from Streptomyces lividans TK24, resulting in RedStrep strains with dramatically increased heterologous production of mithramycin A (up to 3-g/l culture). This system, however, required a time-consuming step to remove the resistance marker genes. In order to simplify markerless deletions, we prepared a new system based on the plasmid pAMR18A. This plasmid contains a large polylinker with many unique restriction sites flanked by apramycin and kanamycin resistance genes and the bpsA gene for selecting a double-crossover event. The utility of this new markerless deletion system was demonstrated by its deletion of a 21-kb actinorhodin gene cluster from Streptomyces lividans TK24 with 30% efficiency. We used this system to efficiently remove the matA and matB genes in selected RedStrep strains, resulting in biotechnologically improved strains with a highly dispersed growth phenotype involving non-pelleting small and open mycelia. No further increase in mithramycin A production was observed in these new RedStrep strains, however. We also used this system for the markerless insertion of a heterologous mCherry gene, an improved variant of the monomeric red fluorescent protein, under the control of the strong secretory signal sequence of the subtilisin inhibitor protein, into the chromosome of S. lividans TK24. The resulting recombinant strains efficiently secreted mCherry into the growth medium in a yield of 30 mg/l.

Topics: Amino Acid Sequence; Anthraquinones; Bacterial Proteins; Chromosomes, Bacterial; DNA, Bacterial; Gene Deletion; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genetic Markers; Industrial Microbiology; Multigene Family; Piperidones; Plasmids; Plicamycin; Streptomyces; Streptomyces lividans