“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.
NFAT-133 is a Streptomyces-derived aromatic polyketide compound with immunosuppressive, antidiabetic, and antitrypanosomal activities. It inhibits transcription mediated by nuclear factor of activated T cells (NFAT), leading to the suppression of interleukin-2 expression and T cell proliferation. It also activates the AMPK pathway in L6 myotubes and increases glucose uptake. In addition to NFAT-133, a number of its congeners, e.g., panowamycins and benwamycins, have been identified. However, little is known about their modes of formation in the producing organisms. Through genome sequencing of Streptomyces pactum ATCC 27456, gene inactivation, and genetic complementation experiments, the biosynthetic gene cluster of NFAT-133 and its congeners has been identified. The cluster contains a highly disordered genetic organization of type I modular polyketide synthase genes with several genes that are necessary for the formation of the aromatic core unit and tailoring processes. In addition, a number of new analogs of NFAT-133 were isolated and their chemical structures elucidated. It is suggested that the heptaketide NFAT-133 is derived from an octaketide intermediate, TM-123. The current study shows yet another unusual biosynthetic pathway involving a noncanonical polyketide synthase assembly line to produce a group of small molecules with valuable bioactivities.
Nature has always been seemingly limitless in its ability to create new chemical entities. It provides vastly diverse natural compounds through a biomanufacturing process that involves myriads of biosynthetic machineries. Here we report a case of unusual formations of hybrid natural products that are derived from two distinct polyketide biosynthetic pathways, the NFAT-133 and conglobatin pathways, in Streptomyces pactum ATCC 27456. Their chemical structures were determined by NMR spectroscopy, mass spectrometry, and chemical synthesis. Genome sequence analysis and gene inactivation experiments uncovered the biosynthetic gene cluster of conglobatin in S. pactum. Biochemical studies of the recombinant thioesterase (TE) domain of the conglobatin polyketide synthase (PKS) as well as its S74A mutant revealed that the formation of these hybrid compounds requires an active TE domain. We propose that NFAT-133 can interfere with conglobatin biosynthesis by reacting with the TE-domain-bound intermediates in the conglobatin PKS assembly line to form hybrid NFAT-133/conglobatin products.
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...
The soil bacterium Streptomyces pactum ATCC 27456 produces a number of polyketide natural products. Among them is NFAT-133, an inhibitor of the nuclear factor of activated T cells (NFAT) that suppresses interleukin-2 (IL-2) expression and T cell proliferation. Biosynthetic gene inactivation in the ATCC 27456 strain revealed the ability of this strain to produce other polyketide compounds including analogues of NFAT-133. Consequently, seven new derivatives of NFAT-133, TM-129–TM-135, together with a known compound, panowamycin A, were isolated from the culture broth of S. pactum ATCC 27456 ΔptmTDQ. Their chemical structures were elucidated on the basis of their HRESIMS, 1D and 2D NMR spectroscopy, and ECD calculation and spectral data. NFAT-133, TM-132, TM-135, and panowamycin A showed no antibacterial activity or cytotoxicity, but weakly reduced the production of LPS-induced nitric oxide in RAW264.7 cells in a dose-dependent manner. A revised chemical structure of panowamycin A and proposed modes of formation of the new NFAT-133 analogues are also presented.
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