Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism

Abstract

To construct a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis, the genome of the industrial microorganism Streptomyces avermitilis was systematically deleted to remove nonessential genes. A region of more than 1.4 Mb was deleted stepwise from the 9.02-Mb S. avermitilis linear chromosome to generate a series of defined deletion mutants, corresponding to 83.12-81.46% of the wild-type chromosome, that did not produce any of the major endogenous secondary metabolites found in the parent strain. The suitability of the mutants as hosts for efficient production of foreign metabolites was shown by heterologous expression of three different exogenous biosynthetic gene clusters encoding the biosynthesis of streptomycin (from S. griseus Institute for Fermentation, Osaka [IFO] 13350), cephamycin C (from S. clavuligerus American type culture collection (ATCC) 27064), and pladienolide (from S. platensis Mer-11107). Both streptomycin and cephamycin C were efficiently produced by individual transformants at levels higher than those of the native-producing species. Although pladienolide was not produced by a deletion mutant transformed with the corresponding intact biosynthetic gene cluster, production of the macrolide was enabled by introduction of an extra copy of the regulatory gene pldR expressed under control of an alternative promoter. Another mutant optimized for terpenoid production efficiently produced the plant terpenoid intermediate, amorpha-4,11-diene, by introduction of a synthetic gene optimized for Streptomyces codon usage. These findings highlight the strength and flexibility of engineered S. avermitilis as a model host for heterologous gene expression, resulting in the production of exogenous natural and unnatural metabolites.genome engineering | host development | natural products A prominent property of members of the genus Streptomyces is the ability to produce numerous secondary metabolites, including antibiotics and other biologically active compounds of proven value in human and veterinary medicine and agriculture; they are also useful as biochemical tools. These structurally diverse metabolites collectively express not only antibacterial, antifungal, antiviral, and antitumor activities but also antihypertensive and immunosuppressant properties. Streptomyces have been a rich source of pharmaceutical compounds in which common cellular intermediates, including amino acids, sugars, fatty acids, and terpenes, are combined to give more complex structures by defined biochemical pathways. Genomic analysis of three species of Streptomyces, S. avermitilis (1, 2), S. coelicolor A3(2) (3), and S. griseus (4), has revealed that these microorganisms each have large linear chromosomes that harbor over 20 secondary metabolic gene clusters encoding the biosynthesis of polyketides by polyketide synthases (PKSs), peptides by nonribosomal peptide synthetases (NRPSs), bacteriocins, terpenoids, shikimate-derived metabolites, aminoglycosides, and other natural products (5).T...

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