“Trojan horse” antibiotic albomycins are peptidyl nucleosides consisting of a highly modified 4′-thiofuranosyl cytosine moiety and a ferrichrome siderophore that are linked by a peptide bond via a serine residue. While the latter component serves to sequester iron from the environment, the seryl nucleoside portion is a potent inhibitor of bacterial seryl-tRNA synthetases, resulting in broad-spectrum antimicrobial activities of albomycin δ2. The isolation of albomycins has revealed this biological activity is only optimized following two unusual cytosine modifications, N4-carbamoylation and N3-methylation. We identified a genetic locus (named abm) for albomycin production in Streptomyces sp. ATCC 700974. Gene deletion and complementation experiments along with bioinformatic analysis suggested 18 genes are responsible for albomycin biosynthesis and resistance, allowing us to propose a potential biosynthetic pathway for installing the novel chemical features. The gene abmI, encoding a putative methyltransferase, was functionally assigned in vitro and shown to modify the N3 of a variety of cytosine-containing nucleosides and antibiotics such as blasticidin S. Furthermore, a ΔabmI mutant was shown to produce the descarbamoyl-desmethyl albomycin analog, supporting that the N3-methylation occurs before the N4-carbamoylation in the biosynthesis of albomycin δ2. The combined genetic information was utilized to identify an abm-related locus (named ctj) from the draft genome of Streptomyces sp. C. Cross-complementation experiments and in vitro studies with CtjF, the AbmI homolog, suggest the production of a similar 4′-thiofuranosyl cytosine in this organism. In total, the genetic and biochemical data provide a biosynthetic template for assembling siderophore-inhibitor conjugates, and modifying the albomycin scaffold to generate new derivatives.
Nonribosomal peptide synthetases (NRPSs) are involved in the biosynthesis of numerous peptide and peptide-like natural products that have been exploited in medicine, agriculture, and biotechnology, among other fields. As a consequence, there have been considerable efforts aimed at understanding how NRPSs orchestrate the assembly of these natural products. This review highlights several recent examples that continue to expand upon the fundamental knowledge of NRPS mechanism and includes (1) the discovery of new NRPS substrates and the mechanism by which these sometimes structurally complex substrates are made, (2) the characterization of new NRPS activities and domains that function during the process of peptide assembly, and (3) the various catalytic strategies that are utilized to release the NRPS product. These findings continue to strengthen the predictive power for connecting genes-to-products, thereby facilitating natural product discovery and development in the Genomics Era.
Modified nucleosides produced by Streptomyces and related actinomycetes are widely used in agriculture and medicine as antibacterial, antifungal, anticancer and antiviral agents. These specialized small-molecule metabolites are biosynthesized by complex enzymatic machineries encoded within gene clusters in the genome. The past decade has witnessed a burst of reports defining the key metabolic processes involved in the biosynthesis of several distinct families of nucleoside antibiotics. Furthermore, genome sequencing of various Streptomyces species has dramatically increased over recent years. Potential biosynthetic gene clusters for novel nucleoside antibiotics are now apparent by analysis of these genomes. Here we revisit strategies for production improvement of nucleoside antibiotics that have defined mechanisms of action, and are in clinical or agricultural use. We summarize the progress for genetically manipulating biosynthetic pathways for structural diversification of nucleoside antibiotics. Microorganism-based biosynthetic examples are provided and organized under genetic principles and metabolic engineering guidelines. We show perspectives on the future of combinatorial biosynthesis, and present a working model for discovery of novel nucleoside natural products in Streptomyces.
c Albomycin (ABM), also known as grisein, is a sulfur-containing metabolite produced by Streptomyces griseus ATCC 700974. Genes predicted to be involved in the biosynthesis of ABM and ABM-like molecules are found in the genomes of other actinomycetes. ABM has potent antibacterial activity, and as a result, many attempts have been made to develop ABM into a drug since the last century. Although the productivity of S. griseus can be increased with random mutagenesis methods, understanding of Streptomyces sulfur amino acid (SAA) metabolism, which supplies a precursor for ABM biosynthesis, could lead to improved and stable production. We previously characterized the gene cluster (abm) in the genome-sequenced S. griseus strain and proposed that the sulfur atom of ABM is derived from either cysteine (Cys) or homocysteine (Hcy). The gene product, AbmD, appears to be an important link between primary and secondary sulfur metabolic pathways. Here, we show that propargylglycine or iron supplementation in growth media increased ABM production by significantly changing the relative concentrations of intracellular Cys and Hcy. An SAA metabolic network of S. griseus was constructed. Pathways toward increasing Hcy were shown to positively impact ABM production. The abmD gene and five genes that increased the Hcy/Cys ratio were assembled downstream of hrdBp promoter sequences and integrated into the chromosome for overexpression. The ABM titer of one engineered strain, SCAK3, in a chemically defined medium was consistently improved to levels ϳ400% of the wild type. Finally, we analyzed the production and growth of SCAK3 in shake flasks for further process development. The Streptomyces genus was established at the beginning of the golden age of antibiotic discovery (1). Streptomyces griseus, a representative organism of the genus (2), was shown during this time to produce streptomycin (3), which has been clinically used to treat bacterial infections and other human diseases. It is well known that Streptomyces organisms in general have a large biosynthetic potential, being capable of producing many bioactive secondary metabolites-the S. griseus strains producing albomycin (ABM) are no exception. ABM was named by former Soviet Union scientists and underwent clinical investigations well before the information was released to the English-speaking scientific world (4). The biological activity and chemical constitution of ABM were later reported to be nearly identical to those of grisein, which was isolated from a distinct subtype of S. griseus by U.S. scientists in the 1940s (5-7). Despite its remarkable properties, continued studies of ABM were not pursued, partly because of the poor yields (8) and unpublicized research on the microorganism (9, 10). At present, the wealth of Streptomyces genomic information that is publically available provides the possibility of using a systems biology approach to uncover new aspects of Streptomyces metabolism and potentially overcome the production bottleneck.ABM and ABM-like secondary metabolites...
telephone: (608) 263-2673, and fax: (608) 262-5345The archetypical polyketide synthases (PKSs), known as type I, II, and III PKSs, have accounted for the vast structural diversities embodied by polyketide natural products, but recent progress in polyketide biosynthesis clearly suggests much greater diversity for PKS mechanism and structure. We have previously argued the emergence of novel PKSs and cautioned the oversimplification of polyketide biosynthesis according to the type I, II, and III PKS paradigms on the basis of our studies on the biosynthesis of the enediynes, the macrotetrolides, and leinamycin. We present here a brief progress report on our current effort to mechanistically characterize these novel PKSs. 154
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