New genetic methods to improve secondary metabolite production in Streptomyces

The most potent chemical mutagens commonly used for yield enhancement in streptomycetes induce a limited spectrum of base-pair substitutions, heavily dominated by GC to AT transitions. The AT to CG transversion pathway complements the GC to AT pathway, but no strong mutagens with this specificity are available. However, mutT mutations in Escherichia coli enhance spontaneous AT to CG transversions about 1000-fold, so such mutations in streptomycetes could be very beneficial for random mutagenesis. Rate-limiting steps in secondary metabolite biosynthesis, on the other hand, can be best addressed by cloning and insertion of extra copies of the appropriate gene(s) into a neutral genomic site. This approach has been used successfully to improve the production of tylosin and pristinomycin. Transposon mutagenesis can be used to identify and clone neutral genomic sites, as demonstrated in Streptomyces fradiae to improve tylosin yields. Transposon mutagenesis can also be used to activate or enhance the transcription of genes important for secondary metabolite production, and to identify and clone both positive and negative regulatory elements. In some cases it may be advantageous to amplify the complete biosynthetic pathway for secondary metabolite production. In Streptomyces rimosus, a highly productive oxytetracycline producer was shown to contain the complete biosynthetic pathway near the end of the linear chromosome, and on a hybrid linear plasmid that had undergone recombination with the chromosome. This concept of linear replicon fusion might be applicable to other secondary metabolite pathway genes that are located near one end of the linear chromosome.