Engineered Biosynthesis of Novel Polyketides: actVII and actIV Genes Encode Aromatase and Cyclase Enzymes, Respectively

McDaniel, Robert; Ebert-Khosla, Susanne; Hopwood, David A.; Khosla, Chaitan

doi:10.1021/ja00103a001

Cited by 105 publications

(123 citation statements)

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“…The respective transformant strains were grown in liquid cultures. All pWEC769 derivatives expressing the S1-, S2-, U1-, and U2-KS ␤ genes (pWEC770 to pWEC773) produced a UV-fluorescent yellow compound and two UV-dark compounds with R f values identical to those of UWM1 (60) and SEK4 and SEK4b (21,43), respectively, when tested by TLC against UWM1, SEK4, and SEK4b standards or by HPLC (Fig. 4B and 5).…”

Section: Resultsmentioning

confidence: 99%

“…After evaporation of the solvent and purification on Sephadex LH20, 4.3 mg (80%) of UWM2 could be obtained. The resulting nuclear mag- a Percent similarity was calculated as the number of matching residues (consensus length) divided by the sum of mismatching residues, matching residues (consensus length), and gapped residues (gap length) ϫ 100. netic resonance (NMR) spectra of UWM1, UWM2, SEK4, and SEK4b were determined with a Bruker AM300 spectrometer; spectra were referenced internally to the solvent and directly compared to the literature (21,43,60).…”

Section: Methodsmentioning

confidence: 99%

“…It includes two ␤-ketoacyl synthase subunits; KS ␣ , which catalyzes the condensation between the acylthioester species and chain length factor (42) (we and others [37,54] prefer the KS ␤ designation for this gene, albeit without any evidence that the two KS proteins form a heterodimer); and an acyl carrier protein (ACP) that acts as an anchor for the polyketide chain during the various biochemical manipulations. Although the minimal PKS alone is sufficient to produce the basic carbon skeleton of the polyketide chain (21,43,44), additional enzymes, such as cyclases (60, 61) (or their equivalent, aromatases [43]) and ketoreductases, are required to fold and cyclize the poly-␤-ketoacyl chain into a (poly)cyclic structure. The challenge in generating novel polyketides is to create a variety of combinations of a minimal PKS, a ketoreductase, and an aromatase or a cyclase to assemble and process nascent polyketides of various chain lengths into a range of novel metabolites.…”

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A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms

et al. 1997

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To examine as randomly as possible the role of the ␤-ketoacyl and acyl carrier protein (ACP) components of bacterial type II polyketide synthases (PKSs), homologs of the chain-length-factor (CLF) genes were cloned from the environmental community of microorganisms. With PCR primers derived from conserved regions of known ketosynthase (KS ␣ ) and ACP genes specifying the formation of 16-to 24-carbon polyketides, two CLF (KS ␤ ) genes were cloned from unclassified streptomycetes isolated from the soil, and two were cloned from soil DNA without the prior isolation of the parent microorganism. The sequence and deduced product of each gene were distinct from those of known KS ␤ genes and, by phylogenetic analysis, belonged to antibiotic-producing PKS gene clusters. Hybrid PKS gene cassettes were constructed with each novel KS ␤ gene substituted for the actI-ORF2 or tcmL KS ␤ subunit genes, along with the respective actI-ORF1 or tcmK KS ␣ , tcmM ACP, and tcmN cyclase genes, and were found to produce an octaketide or decaketide product characteristic of the ones known to be made by the heterologous KS ␣ gene partner. Since substantially less than 1% of the microorganisms present in soil are thought to be cultivatable by standard methods, this work demonstrates a potential way to gain access to a more extensive range of microbial molecular diversity and to biosynthetic pathways whose products can be tested for biological applications.The polyketide metabolites are a large group of structurally complex and diverse natural products, many of which are clinically valuable antibiotics or chemotherapeutic agents or have other useful pharmacological activities (48). Polyketides are synthesized by polyketide synthases (PKSs) which, like the related fatty acid synthases, are multifunctional enzyme assemblies that catalyze repeated decarboxylative condensations between enzyme-bound acylthioesters (52). The structural diversity of polyketides is a result of the different numbers and types of acyl units involved, coupled with various possibilities for enzyme-mediated reduction, dehydration, cyclization, and aromatization of the initial ␤-ketoacyl condensation products (52). Manipulation of the sequence or specificity of the PKS enzymes can lead to the production of novel secondary metabolites (28,29,33). The key to further exploitation of this approach to molecular diversity lies in an increased understanding of the genetics and biochemistry of the various PKS systems.Recent progress in the cloning and sequencing of microbial PKS genes has led to the identification of at least two architecturally different types of PKSs (28, 33). Modular type I PKSs, represented by erythromycin A (11, 17), avermectin (41), rapamycin (58), and soraphen (57), are discrete multifunctional enzymes sporting numerous unique active site domains. The domains are grouped into distinct modules that in turn control the sequential addition of acylthioester units to the growing polyketide chain and the subsequent modification of the respective condensation prod...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms

et al. 1997

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“…Recently, the detailed functions of individual enzymes of a number of polyketides have been deciphered by expressing combinations of different PKS genes (11,23,(32)(33)(34)(35). This has demonstrated the possibility of rationally designing new polyketides by genetic engineering.…”

Section: Discussionmentioning

confidence: 99%

Cloning and heterologous expression of the entire gene clusters for PD 116740 from Streptomyces strain WP 4669 and tetrangulol and tetrangomycin from Streptomyces rimosus NRRL 3016

1997

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The genes for the complete pathways for two polycyclic aromatic polyketides of the angucyclinone class have been cloned and heterologously expressed. Genomic DNAs of Streptomyces rimosus NRRL 3016 and Streptomyces strain WP 4669 were partially digested with MboI, and libraries (ca. 40-kb fragments) in Escherichia coli XL1-Blue MR were prepared with the cosmid vector pOJ446. Hybridization with the actI probe from the actinorhodin polyketide synthase genes identified two clusters of polyketide genes from each organism. After transfer of the four clusters to Streptomyces lividans TK24, expression of one cluster from each organism was established through the identification of pathway-specific products by high-performance liquid chromatography with photodiode array detection. Peaks were identified from the S. rimosus cluster (pks RIM-1 ) for tetrangulol, tetrangomycin, and fridamycin E. Peaks were identified from the WP 4669 cluster (pks WP-2 ) for tetrangulol, 19-hydroxytetrangulol, 8-O-methyltetrangulol, 19-hydroxy-8-O-methyltetrangulol, and PD 116740. Structures were confirmed by 1 H nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry.Members of the genus Streptomyces produce well over half of the known antibiotics of natural origin. Of these, polyketidederived metabolites are among the most numerous and diverse and include many clinically important members, such as erythromycin, tetracycline, and doxorubicin. Assembly of the skeletons of such compounds by oligomerization of small precursor fatty acid thioesters to form polyketide backbones is carried out by polyketide synthases (PKSs). These are made up of either large multifunctional enzymes (type I PKSs) or multienzyme complexes (type II PKSs). Molecular genetic analyses have revealed that the genes for each PKS are clustered in a relatively small region of DNA (22). The gene clusters for numerous polyketide pathways have been detected by hybridization with probes derived from genes coding for proteins that catalyze equivalent reactions in different pathways (25,29).Angucyclinones, a name recently given to naturally occurring benz [a]anthraquinones, are a rapidly growing group of polyketide natural products, involving many bioactive compounds (41). They are now the largest class of known aromatic decaketides. Two angucyclinones-tetrangulol (TET) and tetrangomycin-had been isolated from Streptomyces rimosus and were the first identified members of this class of antibiotics (26). Streptomyces strain WP 4669 produces an angucyclinone, PD 116740, which has activity against L1210 lymphocytic leukemia and HCT-8 colon adenocarcinoma cell lines (47). We have studied the biosynthesis of a number of angucyclinonederived metabolites (15, 16, 43), including PD 116740 (14). In these pathways, either dehydrorabelomycin (27, 43) or TET (13, 26), plays a key role (Fig. 1). These studies have revealed that the polyketide assembly common to both of these diverges at a prearomatic stage with an additional ketone reduction (at C-6) leading to TET. We have...

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“…Sequence analysis of the tcmN gene (12) showed that only the N-terminal portion of the deduced amino acid sequence is homologous to other putative polyketide cyclase genes or their closely related aromatases (30), such as the ActVII protein from S. coelicolor (31). The latter type of proteins exhibits an internal "duplication" motif in which the sequences of the Nand C-terminal halves of the protein are similar (32).…”

Section: Methodsmentioning

confidence: 99%

Deciphering the mechanism for the assembly of aromatic polyketides by a bacterial polyketide synthase.

Shen

Hutchinson

1996

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

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Aromatic polyketides are assembled by a type II (iterative) polyketide synthase (PKS) in bacteria. Understanding the enzymology of such enzymes should provide the information needed for the synthesis of novel polyketides through the genetic engineering of PKSs. Using a previously described cell-free system [B.S. & C.R.H. (1993) Polyketide metabolites are one of the largest groups of natural products, many of which are clinically valuable antibiotics or chemotherapeutic agents, or exhibit other pharmacological activities (1, 2). Despite their apparent structural diversity, they share a common mechanism of biosynthesis. The carbon backbone of a polyketide results from sequential condensation of short fatty acids, such as acetate, propionate, or butyrate, in a manner resembling fatty acid biosynthesis but is catalyzed by polyketide synthases (PKSs). A strong sequence and mechanistic similarity among many of the PKSs has led to two paradigms for explaining polyketide biochemistry: type I PKSs, multifunctional proteins that harbor a distinct active site for every enzyme-catalyzed step, and type II PKSs, multienzyme complexes that carry a single set of iteratively used activities and consist of several largely monofunctional proteins for the synthesis of complex largely reduced polyketides, like macrolide antibiotics, or aromatic polyketides, like tetracyclines (3, 4, 5). Studies of polyketide biosynthesis provide an excellent model for elucidating the structure-function relationship of complex multienzyme systems, and engineered PKSs have considerable potential for synthesizing novel polyketides (6-9).So far, the examination of type II PKS genes in vivo has revealed limited information about how type II PKSs control product structure, in part because the active sites for the RCOSEnz and CH2(CO2H)COSEnz condensation and oligoketide cyclization reactions are used more than once and, therefore, must recognize different substrates in each bondforming event, as exemplified by the tetracenomycin (Tcm) PKS from Streptomyces glaucescens (see Fig. 1) (10-12). We have previously described a cell-free system to facilitate the purification and reconstitution of individual components of type II PKSs (11), using the Tcm PKS as a model. That work showed that the Tcm PKS consists of the TcmJKLMN proteins (10,(12)(13)(14), although components of a S. glaucescens fatty acid synthase may also be required (11,15). We now report the purification of the TcmN polyketide cyclase and its interaction with the other Tcm PKS proteins (i.e., TcmJKLM), which reveals important mechanistic details about how a type II PKS assembles aromatic polyketides. MATERIALS AND METHODSExpression oftcmN in Streptomyces lividans and Purification of TcmN. A 0.5-kb fragment containing the ermE* promoter was subcloned from pWHM862 (16) into the EcoRI/SstI sites of pGEM3zf (Promega) to give pWHM79. The tcmN gene was then moved as a 1.8-kb BglII/SphI fragment from pWHM862 into the BamHI/SphI sites of pWHM79 to give pWHM80, from which a 2.3-kb BamHI/EcoRI fr...

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Engineered Biosynthesis of Novel Polyketides: actVII and actIV Genes Encode Aromatase and Cyclase Enzymes, Respectively

Cited by 105 publications

References 2 publications

A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms

A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms

Cloning and heterologous expression of the entire gene clusters for PD 116740 from Streptomyces strain WP 4669 and tetrangulol and tetrangomycin from Streptomyces rimosus NRRL 3016

Deciphering the mechanism for the assembly of aromatic polyketides by a bacterial polyketide synthase.

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