Cloning, Nucleotide Sequence, and Heterologous Expression of the Biosynthetic Gene Cluster for R1128, a Non-steroidal Estrogen Receptor Antagonist

Marti, Thomas; Hu, Zhihao; Pohl, Nicola L. B.; Shah, Aseema N.; Khosla, Chaitan

doi:10.1074/jbc.m006766200

Cited by 85 publications

(99 citation statements)

References 45 publications

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“…Examples include enterocin, which is primed by benzoate (13), benastatin, which is primed with hexanoate (14), the R1128 antibiotics, which are primed by a range of short-chain carboxylic acids (15), oxytetracycline, which is primed by malonamate (16), and daunorubicin, which is primed by propionate (17,18). This priming is catalyzed by initiation PKS modules, which vary depending on the priming unit.…”

Section: Resultsmentioning

confidence: 99%

Evolution of polyketide synthases in bacteria

Ridley

Lee

Khosla

2008

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

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165

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The emergence of resistant strains of human pathogens to current antibiotics, along with the demonstrated ability of polyketides as antimicrobial agents, provides strong motivation for understanding how polyketide antibiotics have evolved and diversified in nature. Insights into how bacterial polyketide synthases (PKSs) acquire new metabolic capabilities can guide future laboratory efforts in generating the next generation of polyketide antibiotics. Here, we examine phylogenetic and structural evidence to glean answers to two general questions regarding PKS evolution. How did the exceptionally diverse chemistry of present-day PKSs evolve? And what are the take-home messages for the biosynthetic engineer?biosynthesis ͉ metabolism ͉ engineering P olyketides are a large family of medicinally important natural products, which are formed through the condensation of acylthioester units such as malonyl-CoA and methylmalonyl-CoA to yield metabolites with diverse structures and biological activities. Broadly speaking, there are three separate types of polyketide synthases (PKSs) recognized in bacteria. Multimodular PKSs consist of one or more large multidomain polypeptides where the growing polyketide chain is sequentially passed from one active site to the next. Depending on the nature of their constituent catalytic domains, these megasynthases generate chemical variety and complexity in a stepwise fashion (reviewed in ref. 1). In contrast, iterative PKSs are comprised of a single set of catalysts that assemble a polyketide of controlled chain length through repetitive use of active sites (reviewed in ref.2). In both cases, the nascent polyketide product is frequently acted on by further tailoring enzymes to generate the antibiotic. A third type of PKS (called type III PKSs) is fundamentally different in that the growing polyketide chain is never directly attached to a protein (reviewed in ref.3). This article focuses on the evolution of only the first two PKS classes.Bacteria, in particular Actinomycetes and Cyanobacteria, are prolific sources of polyketides, many of which possess antibiotic activity. Erythromycin, tetracycline, and amphotericin B are three well known examples of antimicrobial warfare agents from this group of bacteria that have been found useful for treating human diseases. Polyketides have also been discovered that play other roles in the environment other than to defeat microbial competitors. One such polyketide, mycolactone, is a pathogenesis-enabling immunosuppressant produced by the bacterium Mycobacterium ulcerans. This human pathogen is the causative agent of Buruli ulcer, but M. ulcerans mycolactone-negative mutants are avirulent. Addition of mycolactone to the mycolactone-negative mutants (chemical complementation) restores virulence (4). Other polyketides have been discovered that support a symbiotic relationship. One such compound, rhizoxin, is produced by a Burkholderia sp. symbiont of the fungus Rhizopus sp. Without the symbiont, the fungus cannot make the polyketide that functions to inh...

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Section: Resultsmentioning

confidence: 99%

Evolution of polyketide synthases in bacteria

Ridley

Lee

Khosla

2008

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

Self Cite

165

156

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“…Many other fungal PKSs, however, have a single ACP (15). It was reported in the R1128 anthraquinonelike estrogen receptor antagonist gene cluster that the presence of two acyl carrier proteins is because of the two sets of ketosynthases and acyl transferases with which the ACPs might interact in loading the unusual primer unit (40). The ACPs of R1128 gene cluster are, however, not tandemly repeated and are divergently transcribed unlike mup ACPs.…”

Section: Role Of Tandemly Repeated Acyl Carrier Proteinsmentioning

confidence: 99%

Tandemly Duplicated Acyl Carrier Proteins, Which Increase Polyketide Antibiotic Production, Can Apparently Function Either in Parallel or in Series

Rahman

Hothersall

Crosby

et al. 2005

Journal of Biological Chemistry

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Polyketide biosynthesis involves the addition of subunits commonly derived from malonate or methylmalonate to a starter unit such as acetate. Type I polyketide synthases are multifunctional polypeptides that contain one or more modules, each of which normally contains all the enzymatic domains for a single round of extension and modification of the polyketide backbone. Acyl carrier proteins (ACP(s)) hold the extender unit to which the starter or growing chain is added. Normally there is one ACP for each ketosynthase module. However, there are an increasing number of known examples of tandemly repeated ACP domains, whose function is as yet unknown. For the doublet and triplet ACP domains in the biosynthetic pathway for the antibiotic mupirocin from Pseudomonas fluorescens NCIMB10586 we have inactivated ACP domains by inframe deletion and amino acid substitution of the active site serine. By deletion analysis each individual ACP from a cluster can provide a basic but reduced activity for the pathway. In the doublet cluster, substitution analysis indicates that the pathway may follow two parallel routes, one via each of the ACPs, thus increasing overall pathway flow. In the triplet cluster, substitution in ACP5 blocked the pathway. Thus ACP5 appears to be arranged "in series" to ACP6 and ACP7. Thus although both the doublet and triplet clusters increase antibiotic production, the mechanisms by which they do this appear to be different and depend specifically on the biosynthetic stage involved. The function of some ACPs may be determined by their location in the protein rather than absolute enzymic activity.

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“…While many minimal PKSs use acetate, in the form of acetyl-ACP, to initiate polyketide biosynthesis, a large number of minimal PKSs, such as those from the pathways of enterocin (Piel et al, 2000), R1128 (Marti et al, 2000), doxorubicin (Ye et al, 1994), hedamycin (Bililign et al, 2004;Das & Khosla, 2009) and fredericamycin (Wendt-Pienkowski et al, 2005), have been found to use a variety of non-acetate starter units.…”

Section: Discussionmentioning

confidence: 99%

Genetic characterization of enzymes involved in the priming steps of oxytetracycline biosynthesis in Streptomyces rimosus

et al. 2011

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Tetracyclines are clinically important aromatic polyketides whose biosynthesis is catalysed by bacterial type II polyketide synthases (PKSs). Tetracyclines are biosynthesized starting with an amide-containing malonamate starter unit and the resulting C-2 carboxyamide is critical for the antibiotic activities. In this work, we genetically verified that an amidotransferase, OxyD, and a thiolase, OxyP, are involved in the biosynthesis and incorporation of the starter unit. First, two mutations, R248T and D268N, were found to be present in OxyD* encoded in Streptomyces rimosus ATCC 13224, a strain that produces the acetate-primed 2-acetyl-2-decarboxyamido-oxytetracycline (ADOTC) instead of the malonamate-primed oxytetracycline (OTC). Homology modelling suggested that in particular D268N may inactivate OxyD. Complementation of S. rimosus ATCC 13224 with wild-type OxyD restored OTC biosynthesis, thereby confirming the essential role of OxyD in the synthesis of the amide starter unit. Second, using a series of knockout and complementation approaches, we demonstrated that OxyP is most likely involved in maintaining fidelity of the amide-priming process via hydrolysis of the competing acetate priming starter units. While the inactivation of OxyP does not eliminate OTC biosynthesis, the ratio of acetate-primed ADOTC to malonamate-primed OTC is significantly increased. This suggests that OxyP plays an ancillary role in OTC biosynthesis and is important for minimizing the levels of ADOTC, a shunt product that has much weaker antibiotic activities than OTC.

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Cloning, Nucleotide Sequence, and Heterologous Expression of the Biosynthetic Gene Cluster for R1128, a Non-steroidal Estrogen Receptor Antagonist

Cited by 85 publications

References 45 publications

Evolution of polyketide synthases in bacteria

Evolution of polyketide synthases in bacteria

Tandemly Duplicated Acyl Carrier Proteins, Which Increase Polyketide Antibiotic Production, Can Apparently Function Either in Parallel or in Series

Genetic characterization of enzymes involved in the priming steps of oxytetracycline biosynthesis in Streptomyces rimosus

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