2021 Volume 74 Issue 2 Pages 139-172
After analysis of gene cluster for avermectin (used as a group of antiparasitic agents in human and veterinary medicine) biosynthesis was completed, we got the chance to analyze the genome of avermectin-producing Streptomyces avermitilis. Species of the genus Streptomyces(order Actinomycetales) of major pharmaceutical interest because they synthesized a variety of bioactive secondary metabolites. The linear chromosome of S. avermitilis contains 9,025,608 bases and 7,574 potential open reading frames. Furthermore, more than thirty gene clusters related to secondary metabolite biosynthesis were identified, but almost gene clusters were cryptic state. Comparison with a couple of Streptomyces genomes revealed that an internal 6.5-Mb region in the S. avermitilis genome was highly conserved with respect to gene order and content, and contained all known essential genes, but the terminal regions in both ends were not conserved and preferentially contained nonessential genes, including genes concerning secondary metabolite biosynthesis. In consideration of the genome information, S. avermitilis was constructed as a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis. More than forty of the entire biosynthetic gene clusters for secondary metabolites were successively cloned and introduced into an engineered versatile host of S. avermitilis SUKA (Special Use of Kitasato Actinomytales) series. The production of metabolites in some transformants containing exogenous biosynthetic gene clusters was higher than that of the original producers, and some cryptic biosynthetic gene clusters in the original producer were also expressed in an engineered host SUKA strains. By using our versatile host and bioinformatics of enzymes, many terpene synthases were discovered from bacterial origin and 13 previously unidentified cyclic sesquiterpenes and diterpenes were isolated. Our innovative system concerning an engineered host and a couple of useful vectors was applied to create a new technology for the derivatization of peptide products (ribosomally synthesized and modified by post-translationally) and polyketide compounds synthesized by type I modular polyketide synthases (PKSs). Heterologous expression of many constructs in an engineered host SUKA strain gave 35 designed prethioviridamide derivatives, along with several unprecedented analogues. Cytotoxicity assay revealed that several derivatives showed more potent activities than those of natural product. The strategy can become one of the potential ways to produce supreme unnatural products. Although intense studies have established various methodologies for protein engineering of type I modular PKSs, the accurate targeting of desired regions in the PKS gene is still challenging due to the high sequence similarity between its modules. We developed an innovative technique to edit a target region of the gene encoding type I modular PKS. Proof-of-concept experiments using rapamycin PKS as a template showed that heterologous expression of edited biosynthetic gene clusters produced almost all the desired derivatives. Our technique will provide a platform to generate rationally designed natural product derivatives for future drug development.
