Identification of a Set of Genes Involved in the Formation of the Substrate for the Incorporation of the Unusual “Glycolate” Chain Extension Unit in Ansamitocin Biosynthesis

Carroll, Brian; Moss, Steven J.; Bai, Linquan; Kato, Yasuo; Toelzer, S.; Yu, Tianyi; Floss, Heinz G.

doi:10.1021/ja0124764

Cited by 64 publications

(119 citation statements)

References 18 publications

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“…The 1 H-NMR, 13 C-NMR and MS data were identical with those of compound 3 [20] and were consistent with the molecular formula C 28 reported by Hong et al [20].…”

Section: Structural Elucidation Of Gdmct-1-7 and Gdmct-1-1supporting

confidence: 88%

A New Post-PKS Modification Process in the Carbamoyltransferase Gene Inactivation Strain of Streptomyces hygroscopicus 17997

Wang

et al. 2008

J Antibiot

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Genetic manipulation of geldanamycin (GDM) producer Streptomyces species is a rational approach to understand biosynthesis processes and create new analogues. In this study, the carbamoyltransferase gene gdmN was inactivated by insertion of an apramycinresistance gene aac3 (IV) into the genome of the geldanamycin-producing strain Streptomyces hygroscopicus 17997. GDM analogues produced by this mutant strain were isolated and characterized, such as new compound 4,5-dihydro-7-O -descarbamoyl-7-hydroxy-19-Oglycylgeldanamycin. This compound could be converted to compound 4,5-dihydro-19-O-glycylgeldanamycin, another new GDM analogue, by a strain of Streptomyces hygroscopicus 17997 in which the GDM-pks was inactivated. These new compounds exhibited reductions of cytotoxicity against HepG2 cancer cells, but increases of aqueous solubility. These results suggest that a new postpolyketide synthase modification was involved in this process to produce new GDM analogues.

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“…The 1 H-NMR, 13 C-NMR and MS data were identical with those of compound 3 [20] and were consistent with the molecular formula C 28 reported by Hong et al [20].…”

Section: Structural Elucidation Of Gdmct-1-7 and Gdmct-1-1supporting

confidence: 88%

A New Post-PKS Modification Process in the Carbamoyltransferase Gene Inactivation Strain of Streptomyces hygroscopicus 17997

Wang

et al. 2008

J Antibiot

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“…Based on the available evidence, it is proposed that the substrate for this unusual chainextension reaction is 2-methoxymalonyl-ACP, which is synthesized by the asm13,15-17 gene products on the activated (20) ACP, Asm14. This ACP, which is essential for ansamitocin formation (44), shows greater sequence similarity with ACPs in nonribosomal peptide synthases than in PKSs.…”

Section: Discussionmentioning

confidence: 93%

“…There are also no obvious candidate genes for such a double-bond isomerization outside the PKS region. The possibility that the double-bond migration is related to the presence of the extra oxygen function at C-10 is ruled out by the formation of 10-desmethoxyansamitocin, with the double bonds in the ''abnormal'' position, in the asm15 mutant (44).…”

Section: Discussionmentioning

confidence: 99%

“…With the exception of asm46, which shows no sequence similarity with any known gene, their deduced proteins exhibit high homology (45). A mutant, HGF053, in which the asm15 gene was interrupted by inserting the aac(3)IV gene, showed no ansamitocin production, even in the presence of the side chain precursor, isobutyrate (44), ruling out the involvement of asm15 in the formation of the C-3 ester side chain. However, HPLC, electrospray MS, and NMR analysis revealed the presence of a small amount of 10-desmethoxyansamitocin P-3 in the fermentation of HGF053 (44).…”

Section: Identification and Cloning Of The Ansamitocin Biosynthetic Genementioning

confidence: 99%

“…A mutant, HGF053, in which the asm15 gene was interrupted by inserting the aac(3)IV gene, showed no ansamitocin production, even in the presence of the side chain precursor, isobutyrate (44), ruling out the involvement of asm15 in the formation of the C-3 ester side chain. However, HPLC, electrospray MS, and NMR analysis revealed the presence of a small amount of 10-desmethoxyansamitocin P-3 in the fermentation of HGF053 (44). Therefore, the asm13-16 subcluster, possibly additionally including asm17, may be responsible for generating the ''glycolate'' extender unit, which is incorporated at C-9 and C-10 of ansamitocin.…”

Section: Identification and Cloning Of The Ansamitocin Biosynthetic Genementioning

confidence: 99%

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The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum

Bai

Clade

et al. 2002

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

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280

256

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Maytansinoids are potent antitumor agents found in plants and microorganisms. To elucidate their biosynthesis at the biochemical and genetic level and to set the stage for their structure modification through genetic engineering, we have cloned two gene clusters required for the biosynthesis of the maytansinoid, ansamitocin, from a cosmid library of Actinosynnema pretiosum ssp. auranticum ATCC 31565. This is a rare case in which the genes involved in the formation of a secondary metabolite are dispersed in separate regions in an Actinomycete. A set of genes, asm22-24, asm43-45, and asm47, was identified for the biosynthesis of the starter unit, 3-amino-5-hydroxybenzoic acid (AHBA). Remarkably, there are two AHBA synthase gene homologues, which may have different functions in AHBA formation. Four type I polyketide synthase genes, asmA-D, followed by the downloading asm9, together encode eight homologous sets of enzyme activities (modules), each catalyzing a specific round of chain initiation, elongation, or termination steps, which assemble the ansamitocin polyketide backbone. Another set of genes, asm13-17, encodes the formation of an unusual ''methoxymalonate'' polyketide chain extension unit that, notably, seems to be synthesized on a dedicated acyl carrier protein rather than as a CoA thioester. Additional ORFs are involved in postsynthetic modifications of the initial polyketide synthase product, which include methylations, an epoxidation, an aromatic chlorination, and the introduction of acyl and carbamoyl groups. Tentative functions of several asm genes were confirmed by inactivation and heterologous expression.

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Pellasoren: Struktur, Biosynthese und Totalsynthese eines zytotoxischen Sekundärmetaboliten aus Sorangium cellulosum

et al. 2012

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Auf der Suche nach neuen biologisch aktiven Wirkstoffen hat sich die Untersuchung myxobakterieller Sekundärmetabolite vielfach bewährt. [1][2][3] Hier stellen wir Pellasoren vor, einen weiteren aus dem Myxobakterium Sorangium cellulosum So ce38 isolierten Naturstoff. Neben der kompletten Strukturaufklärung konnten wir durch Identifizierung des pel-Genlocus ferner die Biosynthese des Moleküls erläutern und dabei die Beteiligung einer ungewçhnlichen Glycolat-Verlängerungseinheit postulieren. Die Arbeit umfasst außerdem die erste Totalsynthese von Pellasoren und ermçglicht somit die eindeutige Bestimmung der absoluten Konfiguration dieses Naturstoffs.Pellasoren (1; Schema 1) wurde ursprünglich in einem aktivitätsbasierten Screening in S. cellulosum So ce35 gefunden. [4] Erst kürzlich konnten wir mittels LC-MS-Analysen vergleichsweise große Mengen von 1 in Extrakten des verwandten Stammes S. cellulosum So ce38 nachweisen. Aktivitätstests zeigten mit einem IC50-Wert von 155 nm eine nennenswerte zytotoxische Aktivität gegen Darmkrebszellen der Linie HCT-116. Parallel dazu erfolgte die Aufklärung der Struktur durch NMR-und hrESI-MS-Messungen, wobei die Identität des Pellasorens in beiden Stämmen zweifelsfrei nachgewiesen wurde (siehe Hintergrundinformationen, Abbildungen S.1-3 und Tabelle S.1).Die Struktur des Pellasorens weist eine für Naturstoffe seltene Enolether-Funktionalität auf, die nur für wenige Naturstoffe dokumentiert wurde. HMBC-Kopplungen zwischen einem Methoxysignal und einem sp 2 -hybridisierten Kohlenstoff geben hierfür eindeutige Hinweise (Abbildung S.1). [5][6][7] Das d-Lacton konnte ebenfalls anhand einer HMBC-Kopplung zwischen C1 und C5 sowie durch charakteristische Verschiebungen von 4.02 ppm für H-5 und 90.3 ppm für C5 identifiziert werden. Die Position der Amidbindung wurde durch Analyse der Fragmente in CID-MS 2 -Spektren untermauert, während die relative Konfiguration des Moleküls durch NOE-Spektroskopie und theoretische Studien postuliert wurde. Basierend darauf liegt eine anti-Konfiguration der C4-und C5-Substituenten sowie eine syn-Konfiguration der Methylgruppen an C2 und C4 vor (Abbildung S.2). Die Konfiguration an C14 wiederum beruht auf dem biosynthetischen Einbau eines l-Alanins und wird entsprechend beibehalten, insbesondere da keine Epimerisierungsdomäne im Modul vorliegt. Die Konfiguration des C6-Atoms konnte letztlich nur durch die Totalsynthese bestimmt werden. Es wurde außerdem ein weiteres Derivat des Pellasorens gefunden, das sich lediglich in der Konfiguration der C10-C11-Doppelbindung unterscheidet. Pellasoren A (1 a) entspricht einer all-E-Konfiguration, während Pellasoren B (1 b), hçchstwahrscheinlich beruhend auf einer photochemisch induzierten Isomerisierung, eine (Z)-C10-C11-Konfiguration aufweist.Aufgrund der Struktur konnten wir annehmen, dass Pellasoren ein PKS-NRPS-Hybrid ist, mit Alanin als einzigem Aminosäurebaustein. [8] Um die biochemische Grundlage der Pellasorenbiosynthese zu ermitteln, wurden zunächst alle potentiellen PKS/NRPS-Domänen im Genom des sequenzierten...

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Identification of a Set of Genes Involved in the Formation of the Substrate for the Incorporation of the Unusual “Glycolate” Chain Extension Unit in Ansamitocin Biosynthesis

Cited by 64 publications

References 18 publications

A New Post-PKS Modification Process in the Carbamoyltransferase Gene Inactivation Strain of Streptomyces hygroscopicus 17997

A New Post-PKS Modification Process in the Carbamoyltransferase Gene Inactivation Strain of Streptomyces hygroscopicus 17997

The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum

Pellasoren: Struktur, Biosynthese und Totalsynthese eines zytotoxischen Sekundärmetaboliten aus Sorangium cellulosum

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