S114: Development of genetic systems for engineering and expression of secondary metabolite biosynthetic genes in actinomycetes: issues, challenges, and solutions

Development of genetic systems in microbes often entails: (i) cloning of genes; (ii) introduction and (iii) stable maintenance of DNA in cells; (iv) expression of cloned DNA; (v) engineering of cloned DNA; and (vi) enhancing expression of engineered metabolic pathways. For actinomycetes, the main applications are combinatorial biosynthesis, strain improvement, and expression of cryptic secondary metabolite gene clusters (SMGCs) identified in genome mining programs. Major challenges are: (i) SMGCs are often very large (>50 kb); (ii) it is desirable to have broadly applicable methods to introduce cloned genes into different strains with unpredictable restriction systems; (iii) manufacturing scale up requires maintenance of cloned genes without antibiotic selection; (iv) SMGC genes need to be expressed at appropriate levels; (v) engineering of SMGCs often encounters a paucity of convenient restriction sites; and (vi) strains inevitably need further genetic manipulation to enhance production yields. For actinomycetes, the primary solutions for these challenges have been: (i) use of bacterial artificial chromosome (BAC) vectors to clone large SMGCs; (ii) use of conjugation from Escherichia coli to transfer cloned DNA into a variety of strains, including those that express potent restriction systems; (iii) use of site-specific integration systems to stably insert DNA into chromosomes; (iv) use of the ermE* promoter to drive expression;  (v) use of lambda Red recombination for rapid construction of recombinant pathways; and (vi) use of combinations of methods to enhance expression, including manipulation of positive and negative regulatory genes; selection for resistance to certain antibiotics; and heterologous expression in production hosts.