Solving the Puzzle of One‐Carbon Loss in Ripostatin Biosynthesis

Ripostatin is a promising antibiotic that inhibits RNA polymerase by binding to a novel binding site. In this study, the characterization of the biosynthetic gene cluster of ripostatin, which is a peculiar polyketide synthase (PKS) hybrid cluster encoding cis‐ and trans‐acyltransferase PKS genes, is reported. Moreover, an unprecedented mechanism for phenyl acetic acid formation and loading as a starter unit was discovered. This phenyl‐C2 unit is derived from phenylpyruvate (phenyl‐C3) and the mechanism described herein explains the mysterious loss of one carbon atom in ripostatin biosynthesis from the phenyl‐C3 precursor. Through in vitro reconstitution of the whole loading process, a pyruvate dehydrogenase like protein complex was revealed that performs thiamine pyrophosphate dependent decarboxylation of phenylpyruvate to form a phenylacetyl‐S ‐acyl carrier protein species, which is supplied to the subsequent biosynthetic assembly line for chain extension to finally yield ripostatin.     

Biosynthesis of structurally unique fungal metabolite GKK1032A(2): indication of novel carbocyclic formation mechanism in polyketide biosynthesis

The biosynthesis of the antitumor agent GKK1032A(2) (1) has been investigated by administration of isotopically labeled ((13)C and (2)H) precursors to Penicillium sp. GKK1032. These studies showed that the backbone of 1 is constructed from l-tyrosine and a nonaketide chain flanked with five methyl groups probably by a polyketide synthase and a nonribosomal peptide synthetase hybrid. On the basis of the oxidation level of the starter unit and unusual 13-membered macroether formation between the tyrosine hydroxy group and the polyketide chain, novel cyclization mechanisms on the formation of a tricarbocyclic system and a macroether have been proposed. Involvement of a similar type of cyclization in the biosynthesis of structurally related metabolites is discussed.     

Cloning and elucidation of the FR901464 gene cluster revealing a complex acyltransferase-less polyketide synthase using glycerate as starter units

FR901464, an antitumor natural product, represents a new class of potent anticancer small molecules targeting spliceosome and inhibiting both splicing and nuclear retention of pre-mRNA. Herein we describe the biosynthetic gene cluster of FR901464, identified by degenerate primer PCR amplification of a gene encoding the 3-hydroxy-3-methylglutaryl-CoA synthase (HCS) postulated to be involved in the biosynthesis of a β-branched polyketide from Pseudomonas sp. No. 2663. This cluster consists of twenty open reading frames (ORFs) and was localized to 93-kb DNA segment, and its involvement in FR901464 biosynthesis was confirmed by gene inactivation and complementation. FR901464 is biosynthesized by a hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS), HCS, and acyltransferases (AT)-less system. The PKS/NRPS modules feature unusual domain organization including multiple domain redundancy, inactivation, and tandem. Biochemical characterization of a glyceryl transferase and an acyl carrier protein (ACP) in the start module revealed that it incorporates D-1,3-bisphosphoglycerate, which is dephosphorylated and transferred to ACP as the starter unit. Furthermore, an oxidative Baeyer-Villiger reaction followed by chain release was postulated to form a pyran moiety. On the basis of in silico analysis and genetic and biochemical evidances, a biosynthetic pathway for FR901464 was proposed, which sets the stage to further investigate the complex PKS biochemically and engineer the biosynthetic machinery for the production of novel analogues.     

Yersiniabactin synthetase: a four-protein assembly line producing the nonribosomal peptide/polyketide hybrid siderophore of Yersinia pestis

Yersiniabactin synthetase comprises four proteins, YbtE, HMWP1, HMWP2, and YbtU, encompassing seventeen functional domains, twelve catalytic and five carrier, to select, activate, and incorporate salicylate, three cysteines, and one malonyl moiety into the iron chelator yersiniabactin (Ybt). In the present study, yersiniabactin has been reconstituted in vitro from the 4 protein assembly line by the use of eight biosynthetic precursors. The rate of one turnover, comprising 22 chemical operations performed by the assembly line to release the completed Ybt molecule, was determined at 1.4 min(-1). During the course of Ybt production, the elongating acyl-S-enzyme chain was shown to transfer across a nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) interprotein interface and then a PKS/NRPS intraprotein interface. This study on the Ybt synthetase assembly line represents the first complete in vitro reconstitution of a nonribosomal peptide/polyketide hybrid system.     

Biochemical and structural characterization of germicidin synthase: analysis of a type III polyketide synthase that employs acyl-ACP as a starter unit donor

Germicidin synthase (Gcs) from Streptomyces coelicolor is a type III polyketide synthase (PKS) with broad substrate flexibility for acyl groups linked through a thioester bond to either coenzyme A (CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted in vitro by coupling Gcs with fatty acid biosynthesis. Since Gcs has broad substrate flexibility, we directly compared the kinetic properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA, suggesting a strong preference toward carrier protein starter unit transfer. The 2.9 ? germicidin synthase crystal structure revealed canonical type III PKS architecture along with an unusual helical bundle of unknown function that appears to extend the dimerization interface. A pair of arginine residues adjacent to the active site affect catalytic activity but not ACP binding. This investigation provides new and surprising information about the interactions between type III PKSs and ACPs that will facilitate the construction of engineered systems for production of novel polyketides.     

Sanglifehrin-cyclophilin interaction: degradation work,synthetic macrocyclic analogues,X-ray crystal structure,and binding data

Sanglifehrin A (SFA) is a novel immunosuppressive natural product isolated from Streptomyces sp. A92-308110. SFA has a very strong affinity for cyclophilin A (IC(50) = 6.9 +/- 0.9 nM) but is structurally different from cyclosporin A (CsA) and exerts its immunosuppressive activity via a novel mechanism. SFA has a complex molecular structure consisting of a 22-membered macrocycle, bearing in position 23 a nine-carbon tether terminated by a highly substituted spirobicyclic moiety. Selective oxidative cleavage of the C(26)=C(27) exocyclic double bond affords the spirolactam containing fragment 1 and macrolide 2. The affinity of 2 for cyclophilin (IC(50) = 29 +/- 2.1 nM) is essentially identical to SFA, which indicates that the interaction between SFA and cyclophilin A is mediated exclusively by the macrocyclic portion of the molecule. This observation was confirmed by the X-ray crystal structure resolved at 2.1 A of cyclophilin A complexed to macrolide 16, a close analogue of 2. The X-ray crystal structure showed that macrolide 16 binds to the same deep hydrophobic pocket of cyclophilin A as CsA. Additional valuable details of the structure-activity relationship were obtained by two different chemical approaches: (1) degradation work on macrolide 2 or (2) synthesis of a library of macrolide analogues using the ring-closing metathesis reaction as the key step. Altogether, it appears that the complex macrocyclic fragment of SFA is a highly optimized combination of multiple functionalities including an (E,E)-diene, a short polypropionate fragment, and an unusual tripeptide unit, which together provide an extremely strong affinity for cyclophilin A.     

Chain initiation on the soraphen-producing modular polyketide synthase from Sorangium cellulosum

Background: Polyketides are structurally diverse natural products with a wide range of useful activities. Bacterial modular polyketide synthases (PKSs) catalyse the production of non-aromatic polyketides using a different set of enzymes for each successive cycle of chain extension. The choice of starter unit is governed by the substrate specificity of a distinct loading module. The unusual loading module of the soraphen modular PKS, from the myxobacterium Sorangium cellulosum, specifies a benzoic acid starter unit. Attempts to design functional hybrid PKSs using this loading module provide a stringent test of our understanding of PKS structure and function, since the order of the domains in the loading and first extension module is non-canonical in the soraphen PKS, and the producing strain is not an actinomycete.Results: We have constructed bimodular PKSs based on DEBS1-TE, a derivative of the erythromycin PKS that contains only extension modules 1 and 2 and a thioesterase (TE) domain, by substituting one or more domains from the soraphen PKS. A hybrid PKS containing the soraphen acyltransferase domain AT1b instead of extension acyltransferase domain AT1 produced triketide lactones lacking a methyl group at C-4, as expected if AT1b catalyses the addition of malonyl-CoA during the first extension cycle on the soraphen PKS. Substitution of the DEBS1-TE loading module AT domain by the soraphen AT1a domain led to the production of 5-phenyl-substituted triketide lactone, as well as the normal products of DEBS1-TE. This 5-phenyl triketide lactone was also the product of a hybrid PKS containing the entire soraphen PKS loading module as well as part of its first extension module. Phenyl-substituted lactone was only produced when measures were simultaneously taken to increase the intracellular supply of benzoyl-CoA in the host strain of Saccharopolyspora erythraea.Conclusions: These results demonstrate that the ability to recruit a benzoate starter unit can be conferred on a modular PKS by the transfer either of a single AT domain, or of multiple domains to produce a chimaeric first extension module, from the soraphen PKS. However, benzoyl-CoA needs to be provided within the cell as a specific precursor. The data also support the respective roles previously assigned to the adjacent AT domains of the soraphen loading/first extension module. Construction of such hybrid actinomycete–myxobacterial enzymes should significantly extend the synthetic repertoire of modular PKSs.     

Identification and analysis of the core biosynthetic machinery of tubulysin, a potent cytotoxin with potential anticancer activity

Myxobacteria are well known for their biosynthetic potential, especially for the production of cytotoxic compounds with potential anticancer activities. The tubulysins are currently in preclinical development. They are produced in very low quantities, and genetic manipulation of producing strains has never been accomplished. We report the development of a mariner-based transposon mutagenesis system for Angiococcus disciformis An d48. Extracts from a library of 1200 mutants were analyzed for the presence of tubulysin by a microscopic cell nucleus fragmentation bioassay. The transposition sites of four tubulysin-negative mutants were identified by vector recovery, which led to the identification and the sequencing of the corresponding core biosynthetic gene locus. Sequence analysis of more than 80,000 bp reveals an unusual multimodular hybrid polyketide synthase/peptide synthetase assembly line with a variety of unprecedented features.     

Extender unit and acyl carrier protein specificity of ketosynthase domains of the 6-deoxyerythronolide B synthase

Polyketide synthases (PKSs) catalyze the production of numerous biologically important natural products via repeated decarboxylative condensation reactions. Modular PKSs, such as the 6-deoxyerythronolide B synthase (DEBS), consist of multiple catalytic modules, each containing a unique set of covalently linked catalytic domains. To better understand the engineering opportunities of these assembly lines, the extender unit and acyl carrier protein (ACP) specificity of keto synthase (KS) domains from modules 3 and 6 of DEBS were analyzed. These studies were undertaken with a newly developed didomain [KS][AT] construct, which lacks its own ACP domain and can therefore be interrogated with homologous or heterologous ACP or acyl-ACP substrates. By substituting the natural methylmalonyl extender unit with a malonyl group, a modest role was demonstrated for the KS in recognition of the nucleophilic substrate. The KS domain from module 3 of DEBS was found to exhibit a distinct ACP-recognition profile from the KS domain of module 6. On the basis of the above kinetic insights, a hybrid module was constructed ([KS3][AT3][KR5][ACP5][TE]) which displayed substrate recognition and elongation capabilities consistent with the natural module 3 protein. Unlike module 3, however, which lacks a ketoreductase (KR) domain, the hybrid module was able to catalyze reduction of the beta-ketothioester product of chain elongation. The high expression level and functionality of this hybrid protein demonstrates the usefulness of kinetic analysis for hybrid module design.     

Assembly line termination in cylindrocyclophane biosynthesis: discovery of an editing type II thioesterase domain in a type I polyketide synthase

The termination step is an important source of structural diversity in polyketide biosynthesis. Most type I polyketide synthase (PKS) assembly lines are terminated by a thioesterase (TE) domain located at the C-terminus of the final module, while other PKS assembly lines lack a terminal TE domain and are instead terminated by a separate enzyme in trans. In cylindrocyclophane biosynthesis, the type I modular PKS assembly line is terminated by a freestanding type III PKS (CylI). Unexpectedly, the final module of the type I PKS (CylH) also possesses a C-terminal TE domain. Unlike typical type I PKSs, the CylH TE domain does not influence assembly line termination by CylI in vitro. Instead, this domain phylogenetically resembles a type II TE and possesses activity consistent with an editing function. This finding may shed light on the evolution of unusual PKS termination logic. In addition, the presence of related type II TE domains in many cryptic type I PKS and nonribosomal peptide synthetase (NRPS) assembly lines has implications for pathway annotation, product prediction, and engineering.     

Functional Characterization and Crystal Structure of the Type II Peptidyl Carrier Protein ColA1a in Collismycins Biosynthesis†

Collismycins (COLs) are antibiotics characterized by a 2,2′‐bipyridine (2,2′‐BP) core composed of a trisubstituted ring A and an unmodified ring B. The 2,2′‐BP core, which possesses metal‐chelating ability and plays key roles in various biological activities of COLs, is biosynthesized by a nonribosomal peptide synthetase (NRPS)‐polyketide synthase (PKS) hybrid machinery. The starter module of the NRPS‐PKS hybrid machinery consists of a type II peptidyl carrier protein (PCP) ColA1a and an adenylation protein ColA1b. We here report the functional characterization of ColA1a and ColA1b in vitro, confirming their functions in selection and loading of picolinic acid (PA), instead of normal amino acid substrates, as the origin of ring B in COLs. The 2.1 Å crystal structure of ColA1a was solved, revealing structural features including the additional helices α1a, α1b and missing helix α3, which may reflect unique interactions of ColA1a with other NRPS‐PKS proteins/domains or substrate. Primary and tertiary structural comparison of ColA1a with other PCPs revealed the structural basis for their typical α‐helical bundle, providing a better understanding of the structural flexibility of PCPs. These results facilitate the starter module engineering for the generation of COL derivatives with ring B modifications in the future.     

Stereospecificity of ketoreductase domains 1 and 2 of the tylactone modular polyketide synthase

Tylactone synthase (TYLS) is a modular polyketide synthase that catalyzes the formation of tylactone (1), the parent aglycone precursor of the macrolide antibiotic tylosin. TYLS modules 1 and 2 are responsible for the generation of antidiketide and triketide intermediates, respectively, each bound to an acyl carrier protein (ACP) domain. Each module harbors a ketoreductase (KR) domain. The stereospecificity of TYLS KR1 and TYLS KR2 has been determined by incubating each of the recombinant ketoreductase domains with reconstituted ketosynthase-acyltransferase [KS][AT] and ACP domains from the 6-deoxyerythronolide B synthase (DEBS) in the presence of the N-acetylcysteamine thioester of syn-(2S,3R)-2-methyl-3-hydroxypentanoate (6), methylmalonyl-CoA, and NADPH resulting in the exclusive formation of the ACP-bound (2R,3R,4S,5R)-2,4-methyl-3,5-dihydroxyhepanoyl triketide, as established by GC-MS analysis of the TMS ether of the derived triketide lactone 7. Both TYLS KR1 and KR2 therefore catalyze the stereospecific reduction of the 2-methyl-3-ketoacyl-ACP substrate from the re-face, with specificity for the reduction of the (2R)-methyl (D) diastereomer. The dehydration that is catalyzed by the dehydratase (DH) domains of TYLS module 2 to give the unsaturated (2E,4S,5R)-2,4-dimethyl-5-hydroxyhept-2-enoyl-ACP2 is therefore a syn elimination of water.     

Investigation of the early stages in soraphen A biosynthesis

The unusual benzoate starter unit in soraphen A derives from phenylalanine via cinnamate in a beta-oxidative (plant-like) pathway; 3-phenyl-3-hydroxypropanoate incorporates directly into soraphen by loading onto module 2 of the PKS and indirectly from the beta-oxidative pathway to generate benzoyl CoA.     

Isolation, structural elucidation, and biosynthesis of 15-norlankamycin derivatives produced by a type-II thioesterase disruptant of Streptomyces rochei

Lankamycin, a 14-membered macrolide antibiotic, contains a 3-hydroxy-2-butyl side chain at C-13. To analyze the function of lkmE, which encodes type-II thioesterase in the lankamycin cluster, we carried out a gene disruption experiment. Disruption of lkmE resulted in a 70% decrease of lankamycin production concomitant with an accumulation of novel lankamycin derivatives (LM-NS01A and LM-NS01B), in which the C-13 side chain is replaced by a 1-carboxyethyl group. The biosynthetic origin of 1-carboxyethyl group was confirmed by incorporation of deuterium in [3-²H]3-methyl-2-oxobutyrate into the C-14 position. These results indicate that the biosynthesis of LM-NS01A and LM-NS01B starts from isobutyryl CoA in place of (S)-2-methylbutyryl CoA and LkmE removes the aberrantly loaded starter unit and restores lankamycin production.     

Chain initiation on the soraphen-producing modular polyketide synthase from Sorangium cellulosum.

BACKGROUND: Polyketides are structurally diverse natural products with a wide range of useful activities. Bacterial modular polyketide synthases (PKSs) catalyse the production of non-aromatic polyketides using a different set of enzymes for each successive cycle of chain extension. The choice of starter unit is governed by the substrate specificity of a distinct loading module. The unusual loading module of the soraphen modular PKS, from the myxobacterium Sorangium cellulosum, specifies a benzoic acid starter unit. Attempts to design functional hybrid PKSs using this loading module provide a stringent test of our understanding of PKS structure and function, since the order of the domains in the loading and first extension module is non-canonical in the soraphen PKS, and the producing strain is not an actinomycete. RESULTS: We have constructed bimodular PKSs based on DEBS1-TE, a derivative of the erythromycin PKS that contains only extension modules 1 and 2 and a thioesterase (TE) domain, by substituting one or more domains from the soraphen PKS. A hybrid PKS containing the soraphen acyltransferase domain AT1b instead of extension acyltransferase domain AT1 produced triketide lactones lacking a methyl group at C-4, as expected if AT1b catalyses the addition of malonyl-CoA during the first extension cycle on the soraphen PKS. Substitution of the DEBS1-TE loading module AT domain by the soraphen AT1a domain led to the production of 5-phenyl-substituted triketide lactone, as well as the normal products of DEBS1-TE. This 5-phenyl triketide lactone was also the product of a hybrid PKS containing the entire soraphen PKS loading module as well as part of its first extension module. Phenyl-substituted lactone was only produced when measures were simultaneously taken to increase the intracellular supply of benzoyl-CoA in the host strain of Saccharopolyspora erythraea. CONCLUSIONS: These results demonstrate that the ability to recruit a benzoate starter unit can be conferred on a modular PKS by the transfer either of a single AT domain, or of multiple domains to produce a chimaeric first extension module, from the soraphen PKS. However, benzoyl-CoA needs to be provided within the cell as a specific precursor. The data also support the respective roles previously assigned to the adjacent AT domains of the soraphen loading/first extension module. Construction of such hybrid actinomycete-myxobacterial enzymes should significantly extend the synthetic repertoire of modular PKSs.     

Two more pieces of the colibactin genotoxin puzzle from Escherichia coli show incorporation of an unusual 1-aminocyclopropanecarboxylic acid moiety

Colibactin represents a structurally undefined class of bacterial genotoxin inducing DNA damage and genomic instability in mammalian cells, thus promoting tumour development and exacerbating lymphopenia in animal models. The colibactin biosynthetic gene cluster (clb) has been known for ten years and it encodes a hybrid nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) assembly line. Nevertheless, the final chemical product(s) remain unknown. Previously, we and others reported several colibactin pathway-related metabolites including N-myristoyl-d-asparagine (1) as part of a prodrug precursor that is cleaved from the putative precolibactin to form active colibactin by the peptidase ClbP. Herein, we report two new colibactin pathway-related metabolites (2 and 3) isolated from a clbP mutant of the probiotic E. coli Nissle 1917 strain. Their structures were established by HRMS and NMR. Compound 2 shows an additional 4-aminopenatanoic acid moiety with respect to 1, while 3 is characterized by the presence of an unusual 7-methyl-4-azaspiro[2.4]hept-6-en-5-one residue. Moreover, we propose the biosynthetic pathway towards both intermediates on the basis of extensive gene inactivation and feeding experiments. The identification of 2 and 3 provides further insight into colibactin biosynthesis including the involvement and formation of a rare 1-aminocyclopropanecarboxylic acid unit. Thus, our work establishes additional steps of the pathway forming the bacterial genotoxin colibactin.     

Sulfonium Acids Loaded onto an Unusual Thiotemplate Assembly Line Construct the Cyclopropanol Warhead of a Burkholderia Virulence Factor

Pathogenic bacteria of the Burkholderia pseudomallei group cause severe infectious diseases such as glanders and melioidosis. Malleicyprols were identified as important bacterial virulence factors, yet the biosynthetic origin of their cyclopropanol warhead has remained enigmatic. By a combination of mutational analysis and metabolomics we found that sulfonium acids, dimethylsulfoniumpropionate (DMSP) and gonyol, known as osmolytes and as crucial components in the global organosulfur cycle, are key intermediates en route to the cyclopropanol unit. Functional genetics and in vitro analyses uncover a specialized pathway to DMSP involving a rare prokaryotic SET‐domain methyltransferase for a cryptic methylation, and show that DMSP is loaded onto the NRPS‐PKS hybrid assembly line by an adenylation domain dedicated to zwitterionic starter units. Then, the megasynthase transforms DMSP into gonyol, as demonstrated by heterologous pathway reconstitution in E. coli.     

Epothilone biosynthesis: assembly of the methylthiazolylcarboxy starter unit on the EpoB subunit

BACKGROUND: Polyketides (PKs) and non-ribosomal peptides (NRPs) are therapeutically important natural products biosynthesized by multimodular protein assembly lines, termed the PK synthases (PKSs) and NRP synthetases (NRPSs), via a similar thiotemplate-mediated mechanism. The potential for productive interaction between these two parallel enzymatic systems has recently been demonstrated, with the discovery that PK/NRP hybrid natural products can be of great therapeutic importance. One newly discovered PK/NRP product, epothilone D from Sorangium cellulosum, has shown great potential as an anti-tumor agent. RESULTS: The chain-initiating methylthiazole ring of epothilone has been generated in vitro as an acyl-S-enzyme intermediate, using five domains from two modules of the polymodular epothilone synthetase. The acyl carrier protein (ACP) domain, excised from the EpoA gene, was expressed in Escherichia coli, purified as an apo protein, and then post-translationally primed with acetyl-CoA using the phosphopantetheinyl transferase enzyme Sfp. The four-domain 150-kDa EpoB subunit (cyclization-adenylation-oxidase-peptidyl carrier protein domains: Cy-A-Ox-PCP) was also expressed and purified in soluble form from E. coli. Post-translational modification with Sfp and CoASH introduced the HS-pantP prosthetic group to the apo-PCP, enabling subsequent loading with L-cysteine to generate the Cys-S-PCP acyl enzyme intermediate. When acetyl-S-ACP (EpoA) and cysteinyl-S-EpoB were mixed, the Cy domain of EpoB catalyzed acetyl transfer from EpoA to the amino group of the Cys-S-EpoB, generating a transient N-Ac-Cys-S-EpoB intermediate that is cyclized and dehydrated to the five-membered ring methylthiazolinyl-S-EpoB. Finally, the FMN-containing Ox domain of EpoB oxidized the dihydro heterocyclic thiazolinyl ring to the heteroaromatic oxidation state, the methylthiazolylcarboxy-S-EpoB. When other acyl-CoAs were substituted for acetyl-CoA in the Sfp-based priming of the apo-CP domain, additional alkylthiazolylcarboxy-S-EpoB acyl enzymes were produced. CONCLUSIONS: These experiments establish chain transfer across a PKS and NRPS interface. Transfer of the acetyl group from the ACP domain of EpoA to EpoB reconstitutes the start of the epothilone synthetase assembly line, and installs and converts a cysteine group into a methyl-substituted heterocycle during this natural product chain growth.     

Iterative Assembly of Two Separate Polyketide Chains by the Same Single‐Module Bacterial Polyketide Synthase in the Biosynthesis of HSAF

Antifungal HSAF (heat‐stable antifungal factor, dihydromaltophilin) is a polycyclic tetramate macrolactam from the biocontrol agent Lysobacter enzymogenes. Its biosynthetic gene cluster contains only a single‐module polyketide synthase–nonribosomal peptide synthetase (PKS‐NRPS), although two separate hexaketide chains are required to assemble the skeleton. To address the unusual biosynthetic mechanism, we expressed the biosynthetic genes in two “clean” strains of Streptomyces and showed the production of HSAF analogues and a polyene tetramate intermediate. We then expressed the PKS module in Escherichia coli and purified the enzyme. Upon incubation of the enzyme with acyl‐coenzyme A and reduced nicotinamide adenine dinucleotide phosphate (NADPH), a polyene was detected in the tryptic acyl carrier protein (ACP). Finally, we incubated the polyene–PKS with the NRPS module in the presence of ornithine and adenosine triphosphate (ATP), and we detected the same polyene tetramate as that in Streptomyces transformed with the PKS‐NRPS alone. Together, our results provide evidence for an unusual iterative biosynthetic mechanism for bacterial polyketide–peptide natural products.     

Identification of caerulomycin A gene cluster implicates a tailoring amidohydrolase

The biosynthetic gene cluster for caerulomycin A (1) was cloned and characterized from the marine actinomycete Actinoalloteichus cyanogriseus WH1-2216-6, which revealed an unusual hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) system. The crmL disruption mutant accumulated caerulomycin L (2) with an extended L-leucine at C-7, implicating an amidohydrolase activity for CrmL. The leucine-removing activity was confirmed for crude CrmL enzymes. Heterologous expression of the 1 gene cluster led to 1 production in Streptomyces coelicolor.