Molecular genetic analysis reveals a putative bifunctional polyketide cyclase/dehydrase gene from Streptomycea coelicolor and Streptomyces violoceoruber, and a cyclase/O-methyltransferase from Streptomyces glaucescens.

Sherman, David H.; Bibb, Maureen J.; Simpson, Thomas J.; Johnson, D. A.; Malpartida, Francisco; Fernández-Moreno, Miguel Ángel; Martínez, E.; Hutchinson, C. Richard; Hopwood, David A.

doi:10.1016/s0040-4020(01)86494-2

Cited by 39 publications

(26 citation statements)

References 46 publications

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“…After completion of successive carbon-carbon condensation which was followed by ketoreduction at the C-9 position, it is believed that the polyketide intermediate can exist as two hypothetical reactive conformations (B and C). It has been shown that structure C is the lower-energy conformer, which supports the idea that mutactin is produced spontaneously by S. coelicolor actVII mutants (32). S. parvulus containing pDHS309 or pDHS310 produced primarily mutactin; however, low levels of aloesaponarin II (as well as of its acidic form) were detected as minor products by GC-MS.…”

Section: Discussionsupporting

confidence: 64%

“…The production of mutactin by S. coelicolor actVII mutants (B40 and B140) has provided valuable insight into the role of actVII-orf4 in type II polyketide chain cyclization (32,33). After completion of successive carbon-carbon condensation which was followed by ketoreduction at the C-9 position, it is believed that the polyketide intermediate can exist as two hypothetical reactive conformations (B and C).…”

Section: Discussionmentioning

confidence: 99%

“…The potential bifunctionality of the actI-orf1 gene product was assessed by generating site-directed mutants in the presumed Cys active site of the condensing enzyme (␤-KS) and Ser active site of the putative acyltransferase (AT) domain (10). In addition, this indicator pathway is used as a quantitative tool to study the roles of the actI-orf2 gene product and of the actVII-orf4-encoded protein (CYC/DH) in mediating intramolecular aldol condensation of the nascent octaketide molecule to two possible cyclization products (32).…”

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Heterologous expression of an engineered biosynthetic pathway: functional dissection of type II polyketide synthase components in Streptomyces species

et al. 1995

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Polyketides are an extensive class of secondary metabolites with diverse molecular structures and biological activities. A plasmid-based multicomponent polyketide synthase expression cassette was constructed using a subset of actinorhodin (act) biosynthetic genes (actI-orf1, actI-orf2, actI-orf3, actIII, actVII, and actIV) from Streptomyces coelicolor which specify the construction of the anthraquinone product aloesaponarin II, a molecule derived from acetyl coenzyme A and 7 malonyl coenzyme A extender units. This system was designed as an indicator pathway in Streptomyces parvulus to quantify polyketide product formation and to examine the functional significance of specific polyketide synthase components, including the act ␤-ketoacyl synthase (␤-KS; encoded by actI-orf1 and actI-orf2) and the act cyclase/dehydrase (encoded by actVII-orf4). Site-directed mutagenesis of the putative active site Cys (to a Gln) in the actI-orf1 ␤-KS product completely abrogated aloesaponarin II production. Changing the putative acyltransferase active-site Ser (to a Leu) located in the actI-orf1 ␤-KS product led to significantly reduced but continued production of aloesaponarin II. Replacement of the expression cassette with one containing a mutant form of actI-orf2 gave no production of aloesaponarin II or any other detectable polyketide products. However, an expression cassette containing a mutant form of actVII-orf4 gave primarily mutactin with low-level production of aloesaponarin II.

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Section: Discussionsupporting

confidence: 64%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Heterologous expression of an engineered biosynthetic pathway: functional dissection of type II polyketide synthase components in Streptomyces species

et al. 1995

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“…Sherman etal., 1989;Bibb etal., 1989;Bergh & Uhlen, 1992), although there is evidence for genes encoding proteins with more than one activity (e.g. Sherman et al, 1991;Summers e t al., 1992). At least in Streptomjces, the corresponding genes involved in the biosynthesis of different polyketides are organized very similarly (Hopwood & Sherman, 1990).…”

Section: Introductionmentioning

confidence: 99%

Identification of a 60 kb region of the chromosome of Pseudomonas fluorescens NCIB 10586 required for the biosynthesis of pseudomonic acid (mupirocin)

et al. 1995

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Pseudomonic acid (mupirocin) produced by Pseudomonas fluorescens is a polyketide antibiotic which blocks isoleucyl-tRNA synthetase. Knowledge of the biosynthetic pathways leading to pseudomonic acid production may help in engineering related antibiotics with other useful properties. To help define these pathways, we have isolated 13 non-producing mutants using Tn5 and Tn1725 mutagenesis. Seven of the Tn5 insertions mapped within a 55 kb region. The remaining insertions, and one gene encoding resistance to pseudomonic acid, mapped at separate locations. DNA that overlapped the seven clustered Tn5 insertions was isolated on a series of clones, extending over a region in excess of 60 kb. Non-producing mutants generated via gene disruption suggest that the biosynthetic cluster extends throughout this region.

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“…ORF2 and ORF3 contain conserved motifs (encoding cysteine and glutamine, respectively) that have been reported as active site motifs in homologous ketosynthase and chain length factors (Bisang et al, 1999). The translated product of ORF4 shows end-to-end similarities with Gra-ORF4 (60 % identity) and ActI-ORF4 (49 % identity ; aromatases) (Sherman et al, 1991 ;Ferna! ndez-Moreno et al, 1992).…”

Section: Cloning and Dna Sequence Of Polyketide Biosynthetic Genesmentioning

confidence: 99%

A polyketide biosynthetic gene cluster from Streptomyces antibioticus includes a LysR-type transcriptional regulator

2001

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In the search for Type II polyketide synthases (PKSs) a DNA fragment was isolated from Streptomyces antibioticus ATCC 11891 (a producer of oleandomycin). DNA sequencing of the cloned fragment revealed six complete ORFs whose deduced products showed similarities to those of other genes known to be involved in polyketide biosynthesis. Several S. coelicolor strains mutated in different steps of actinorhodin biosynthesis (actI, actIII, actV A and actVII) were complemented by the cloned genes, suggesting that the isolated genes encode an aromatic polyketide of unknown structure and function. The cluster also contains a putative LysR-type transcriptional regulator (ORF0), which controls PKS gene expression in a heterologous host. DNA binding assays and transcriptional analysis suggest that the pathway-specific regulator for actinorhodin biosynthesis (actII-ORF4) is also involved in the expression of the cloned PKS in the host strain.

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Molecular genetic analysis reveals a putative bifunctional polyketide cyclase/dehydrase gene from Streptomycea coelicolor and Streptomyces violoceoruber, and a cyclase/O-methyltransferase from Streptomyces glaucescens.

Cited by 39 publications

References 46 publications

Heterologous expression of an engineered biosynthetic pathway: functional dissection of type II polyketide synthase components in Streptomyces species

Heterologous expression of an engineered biosynthetic pathway: functional dissection of type II polyketide synthase components in Streptomyces species

Identification of a 60 kb region of the chromosome of Pseudomonas fluorescens NCIB 10586 required for the biosynthesis of pseudomonic acid (mupirocin)

A polyketide biosynthetic gene cluster from Streptomyces antibioticus includes a LysR-type transcriptional regulator

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