Genome-inspired search for new antibiotics. Isolation and structure determination of new 28-membered polyketide macrolactones, halstoctacosanolides A and B, from Streptomyces halstedii HC34

Tohyama, Shigehiro; Eguchi, Tadashi; Dhakal, Rabindra Prasad; Akashi, Tomoyoshi; Otsuka, Masafumi; Kakinuma, Katsumi

doi:10.1016/j.tet.2004.03.027

Cited by 19 publications

(13 citation statements)

References 27 publications

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“…Consistent with this proposed series of events, when we treated [2-14 C]Mal-S-AcpK and Acac-S-PksL-T2 with PksF, PksG, PksH, and PksI, we observed stable radiolabeling of PksL-T2. In contrast, when the same experiment was performed with [1,[3][4][5][6][7][8][9][10][11][12][13][14] C]Mal-SAcpK, we observed significant loss of radiolabel from PksL-T2 when PksH and PksI were added ( Fig. 5 and Fig.…”

Section: Resultsmentioning

confidence: 70%

“…No decarboxylation was detected when Mal-S-PksL-T2 was incubated with PksF, demonstrating that it is selective for Mal-S-AcpK (data not shown). Treatment of [1,[3][4][5][6][7][8][9][10][11][12][13][14] C]Mal-S-AcpK with PksF gave similar results, with rapid PksF-dependent loss of radiolabel observed, to form [1-14 C]Ac-S-AcpK at a rate of 5.5 M⅐min Ϫ1 (Figs. 8 and 9, which are published as supporting information on the PNAS web site).…”

Section: Resultsmentioning

confidence: 74%

“…Often, the sequence of protein domains and modules is colinear with respect to the sequence of biosynthetic reactions catalyzed by the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) machinery (1)(2)(3). Because of their highly modular nature, the small-molecule products of PKSs, NRPSs, and hybrid NRPS-PKS systems can often be predicted by bioinformatic approaches, and there have been recent experimental validations of such predictions (4)(5)(6). However, for many cases in which the biosynthetic product encoded by a given gene cluster is unknown, functional characterization of the synthase by bioinformatic methods alone is difficult or impossible.…”

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confidence: 99%

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Convergence of isoprene and polyketide biosynthetic machinery: Isoprenyl- S -carrier proteins in the pksX pathway of Bacillus subtilis

Calderone

Kowtoniuk

Kelleher

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

175

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The pksX gene cluster from Bacillus subtilis is predicted to encode the biosynthesis of an as yet uncharacterized hybrid nonribosomal peptide͞polyketide secondary metabolite. We used a combination of biochemical and mass spectrometric techniques to assign functional roles to the proteins AcpK, PksC, PksL, PksF, PksG, PksH, and PksI, and we conclude that they act to incorporate an acetate-derived ␤-methyl branch on an acetoacetyl-S-carrier protein and ultimately generate a ⌬ 2 -isoprenyl-S-carrier protein. This work highlights the power of mass spectrometry to elucidate the functions of orphan biosynthetic enzymes, and it details a mechanism by which single-carbon ␤-branches can be inserted into polyketide-like structures. This pathway represents a noncanonical route to the construction of prenyl units and serves as a prototype for the intersection of isoprenoid and polyketide biosynthetic manifolds in other natural product biosynthetic pathways.mass spectrometry ͉ orphan gene cluster ͉ hybrid nonribosomal peptide͞polyketide ͉ polyketide methylation P olyketides and nonribosomal peptides are classes of secondary metabolites that are synthesized by the iterative coupling of malonyl (Mal) derivatives and amino acids, respectively. The catalytic machinery responsible for the biosynthesis of these natural products comprises modular synthases that incorporate, and often subsequently tailor, monomer units into the growing small molecule by a thiotemplated mechanism. Often, the sequence of protein domains and modules is colinear with respect to the sequence of biosynthetic reactions catalyzed by the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) machinery (1-3). Because of their highly modular nature, the small-molecule products of PKSs, NRPSs, and hybrid NRPS-PKS systems can often be predicted by bioinformatic approaches, and there have been recent experimental validations of such predictions (4-6). However, for many cases in which the biosynthetic product encoded by a given gene cluster is unknown, functional characterization of the synthase by bioinformatic methods alone is difficult or impossible.One such orphan cluster for which the product structure is unknown is the hybrid NRPS-PKS pksX cluster from Bacillus subtilis (7-9). Although the pksX cluster has been proposed to encode the biosynthesis of difficidin (7,9,10), recent work has demonstrated that the NRPS portion of the gene cluster is active, disqualifying difficidin, which does not contain any amines, as a candidate for the biosynthetic product of this cluster (11). Long thought to be cryptic, there is now evidence that it is responsible for the biosynthesis of a product that kills streptomycetes (P. D. Straight, M. A. Fischbach, C.T.W., and R. Kolter, unpublished results). Additionally, this cluster does not follow the usual NRPS-PKS colinearity rules because it has only three trans-acting acyltransferases that may load up to 17 of the 20 predicted thiolation domains of the cluster.We were intrigued by a series of predicted ORFs withi...

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Section: Resultsmentioning

confidence: 70%

Section: Resultsmentioning

confidence: 74%

mentioning

confidence: 99%

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Convergence of isoprene and polyketide biosynthetic machinery: Isoprenyl- S -carrier proteins in the pksX pathway of Bacillus subtilis

Calderone

Kowtoniuk

Kelleher

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

175

230

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“…Subsequent isolation and evaluation of these compounds demonstrated that they possess moderate antimicrobial activity against several microorganisms [3]. However, the complete gene cluster had not been cloned.…”

Section: Shigehiro Tohyama Katsumi Kakinuma Tadashi Eguchimentioning

confidence: 99%

“…45 kb DNA sequence had been cloned for the halstoctacosanolide biosynthetic gene and the DNA sequence contain three open reading flames (ORFs) encoding PKS [3]. To identify the complete gene cluster, genomic walking using a S. halstedii HC34 genome library was curried out by standard hybridization method.…”

Section: Cloning and Sequencing Of Complete Halstoctacosanolide Biosymentioning

confidence: 99%

The Complete Biosynthetic Gene Cluster of the 28-Membered Polyketide Macrolactones, Halstoctacosanolides, from Streptomyces halstedii HC34

2006

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Halstoctacoanolides A and B are 28-membered polyketide macrolactones and were isolated from Streptomyces halstedii HC34. The biosynthetic gene cluster (hls cluster) of halstoctacosanolides was completely identified from the genome library of Streptomyces halstedii HC34. DNA sequence analysis of ca. 100 kb region revealed that there were seven type I polyketide synthases (PKSs) and two cytochrome P450 monooxygenases in this cluster. Involvement of the gene cluster in the halstoctacosanolide biosynthesis was demonstrated by the gene disruption of P450 monooxygenase genes. The mutants produced a new deoxygenated halstoctacosanolide derivative, halstoctacosanolide C, which confirmed that the hls gene cluster was essential for the biosynthesis of halstoctacosanolides.

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Artificial Reconstruction of Two Cryptic Angucycline Antibiotic Biosynthetic Pathways

et al. 2007

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Genome-sequencing projects have revealed that Streptomyces bacteria have the genetic potential to produce considerably larger numbers of natural products than can be observed under standard laboratory conditions. Cryptic angucycline-type aromatic polyketide gene clusters are particularly abundant. Sequencing of two such clusters from Streptomyces sp. PGA64 and H021 revealed the presence of several open reading frames that could be involved in processing the basic angucyclic carbon skeleton. The pga gene cluster contains one putative FAD-dependant monooxygenase (pgaE) and a putatively bifunctional monooxygenase/short chain alcohol reductase (pgaM), whereas the cab cluster contains two similar monooxygenases (cabE and cabM) and an independent reductase (cabV). In this study we have reconstructed the biosynthetic pathways for aglycone synthesis by cloning and sequentially expressing the angucycline tailoring genes with genes required for the synthesis of the unmodified angucycline metabolite-UWM6-in Streptomyces lividans TK24. The expression studies unequivocally showed that, after the production of UWM6, the pathways proceed through the action of the similar monooxygenases PgaE and CabE, followed by reactions catalysed by PgaM and CabMV. Analysis of the metabolites produced revealed that addition of pgaE and cabE genes directs both pathways to a known shunt product, rabelomycin, whereas expression of all genes from a given pathway results in the production of the novel angucycline metabolites gaudimycin A and B. However, one of the end products is most probably further modified by endogenous S. lividans TK24 enzymes. These experiments demonstrate that genes that are either inactive or cryptic in their native host can be used as biosynthetic tools to generate new compounds.

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Genome-inspired search for new antibiotics. Isolation and structure determination of new 28-membered polyketide macrolactones, halstoctacosanolides A and B, from Streptomyces halstedii HC34

Cited by 19 publications

References 27 publications

Convergence of isoprene and polyketide biosynthetic machinery: Isoprenyl- S -carrier proteins in the pksX pathway of Bacillus subtilis

Convergence of isoprene and polyketide biosynthetic machinery: Isoprenyl- S -carrier proteins in the pksX pathway of Bacillus subtilis

The Complete Biosynthetic Gene Cluster of the 28-Membered Polyketide Macrolactones, Halstoctacosanolides, from Streptomyces halstedii HC34

Artificial Reconstruction of Two Cryptic Angucycline Antibiotic Biosynthetic Pathways

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