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Every polyketide synthase module has an acyl carrier protein (ACP) and a ketosynthase (KS) domain that collaborate to catalyze chain elongation. The same ACP then engages the KS domain of the next module to facilitate chain transfer. Understanding the mechanism for this orderly progress of the growing polyketide chain represents a fundamental challenge in assembly line enzymology. Using both experimental and computational approaches, the molecular basis for KS--ACP interactions in the 6-deoxyerythronolide B synthase has been decoded. Surprisingly, KS--ACP recognition is controlled at different interfaces during chain elongation versus chain transfer. In fact, chain elongation is controlled at a docking site remote from the catalytic center. Not only do our findings reveal a new principle in the modular control of polyketide antibiotic biosynthesis, they also provide a rationale for the mandatory homodimeric structure of polyketide synthases, in contrast to the monomeric nonribosomal peptide synthetases. assembly line enzymes | polyketide synthase | protein--protein interactions doi/ 10.1073/pnas.1014081107

The human pathogen Mycobacterium ulcerans produces a polyketide metabolite called mycolactone with potent immunomodulatory activity. M. ulcerans strain Agy99 has a 174 kb plasmid called pMUM001 with three large genes (mlsA1, 51 kb; mlsA2, 7.2 kb; mlsB, 43 kb) that encode type I polyketide synthases (PKS) required for the biosynthesis of mycolactone, as demonstrated by transposon mutagenesis. However, there have been no reports of transfer of the mls locus to another mycobacterium to demonstrate that these genes are sufficient for mycolactone production because in addition to their large size, the mls genes contain a high level of internal sequence repetition, such that the entire 102 kb locus is composed of only 9.5 kb of unique DNA. The combination of their large size and lack of stability during laboratory passage makes them a challenging prospect for transfer to a more rapidly growing and genetically tractable host. Here we describe the construction of two bacterial artificial chromosome Escherichia coli/ Mycobacterium shuttle vectors, one based on the pMUM001 origin of replication bearing mlsB, and the other based on the mycobacteriophage L5 integrase, bearing mlsA1 and m/sA2. The combination of these two constructs permitted the two-step transfer of the entire 174 kb pMUM001 plasmid to Mycobacterium marinum, a rapidly growing non-mycolactone-producing mycobacterium that is a close genetic relative of M. ulcerans. To improve the stability of the mls locus in M. marinum, recA was inactivated by insertion of a hygromycin-resistance gene using double-crossover allelic exchange. As expected, the ArecA mutant displayed increased susceptibility to UV killing and a decreased frequency of homologous recombination. Southern hybridization and RT-PCR confirmed the stable transfer and expression of the m/s genes in both wild-type M. marinum and the recA mutant. However, neither mycolactone nor its predicted precursor metabolites were detected in either strain. These experiments show that it is possible to successfully manipulate and stably transfer the large mls genes, but that other bacterial host factors appear to be required to facilitate mycolactone production.

Phthiocerol dimycocerosates (DIM) and phenolglycolipids (PGL) are functionally important surface-exposed lipids of Mycobacterium tuberculosis. Their biosynthesis involves the products of several genes clustered in a 70-kb region of the M. tuberculosis chromosome. Among these products is PpsD, one of the modular type I polyketide synthases responsible for the synthesis of the lipid core common to DIM and PGL. Bioinformatic analyses have suggested that this protein lacks a functional enoyl reductase activity domain required for the synthesis of these lipids. We have identified a gene, Rv2953, that putatively encodes an enoyl reductase. Mutation in Rv2953 prevents conventional DIM formation and leads to the accumulation of a novel DIM-like product. This product is unsaturated between C-4 and C-5 of phthiocerol. Consistently, complementation of the mutant with a functional pksl5/1 gene from Mycobacterium boris BCG resulted in the accumulation of an unsaturated PGL-like substance. When an intact Rv2953 gene was reintroduced into the mutant strain, the phenotype reverted to the wild type. These findings indicate that Rv2953 encodes a trans-acting enoyl reductase that acts with PpsD in phthiocerol and phenolphthiocerol biosynthesis.

Recent fermentation studies have identified actinomycetes of the marine-dwelling genus Salinispora as prolific natural product producers. To further evaluate their biosynthetic potential, we sequenced the 5,183,331-bp S. tropica CNB-440 circular genome and analyzed all identifiable secondary natural product gene clusters. Our analysis shows that S. tropica dedicates a large percentage of its genome ([approximately equal to] 9.9%) to natural product assembly, which is greater than previous Streptomyces genome sequences as well as other natural product-producing actinomycetes. The S. tropica genome features polyketide synthase systems of every known formally classified family, nonribosomal peptide synthetases, and several hybrid clusters. Although a few clusters appear to encode molecules previously identified in Streptomyces species, the majority of the 17 biosynthetic loci are novel. Specific chemical information about putative and observed natural product molecules is presented and discussed. In addition, our bioinformatic analysis not only was critical for the structure elucidation of the polyene macrolactam salinilactam A, but its structural analysis aided the genome assembly of the highly repetitive sire loci. This study firmly establishes the genus Salinispora as a rich source of drug-like molecules and importantly reveals the powerful interplay between genomic analysis and traditional natural product isolation studies. genome mining I marine bacteria | natural products | polyketide synthase

A photoreactive acetogenin is synthesized. The results unequivocally reveal that the ND1 subunit constructs the inhibitor binding domain, though the contribution of this subunit is challenged.

Aclacinomycin (Acl) oxidoreductase (AknOx) catalyzes the last two steps in the biosynthesis of polyketide antibiotics of the Acl group, the oxidation of the terminal sugar moiety rhodinose to L-aculose. We present the crystal structure of AknOx with bound FAD and the product AclY, refined to 1.65-[Angstrom] resolution. The overall fold of AknOx identifies the enzyme as a member of the p-cresol methylhydroxylase superfamily. The cofactor is bicovalently attached to His-70 and Cys-130 as 8[alpha]-N[delta]1-histidyl, 6-S-cysteinyl FAD. The polyketide ligand is bound in a deep cleft in the substrate--binding domain, with the tetracyclic ring system close to the enzyme surface and the three-sugar chain extending into the protein interior. The terminal sugar residue packs against the isoalloxazine ring of FAD and positions the carbon atoms that are oxidized close to the N5 atom of FAD. The structure and site-directed mutagenesis data presented here are consistent with a mechanism where the two different reactions of AknOx are catalyzed in the same active site but by different active site residues. Tyr-450 is responsible for proton removal from the C-4 hydroxyl group in the first reaction, the oxidation of rhodinose to cinerulose A. Tyr-378 acts as a catalytic base involved in proton abstraction from C3 of cinerulose A in the second reaction, for formation L-aculose. Replacement of this residue, however, does not impair the conversion of rhodinose to cinerulose A. enzyme mechanism | polyketide antibiotic | protein crystallography | protein structure

The diversity of polyketide synthases (PKSs) in sponge-associated microbial communities is analyzed to study the uncultured sponge bacteria. The results have proposed a simplified strategy for the targeted isolation of unusually small PKS genes from complex sponge-symbiont associations.

The bacterial genus Streptomyces has long been appreciated for its ability to produce various kinds of medically important secondary metabolites, such as antibiotics, anti-tumour agents, immunosuppressants and enzyme inhibitors. Tautomycetin (TMC), which is produced by Streptomyces sp. CK4412, is a novel activated T cell-specific immunosuppressive compound with an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. Using a Streptomyces polyketide methylmalonyl-CoA acyltransferase gene as a probe, three overlapping cosmids were isolated from the genomic library of TMC-producing Streptomyces sp. CK4412. Sequence information of an approximately 70 kb contiguous DNA region revealed two multi-modular type I polyketide synthases (PKSs), and 12 additional gene products presumably involved in TMC biosynthesis. The deduced roles for most of the TMC PKS catalytic domains were consistent with the expected functions necessary for TMC chain elongation and processing. In addition, disruption of a putative TMC acyl-CoA transferase gene, located upstream of the PKS gene locus, completely abolished TMC biosynthesis. Taken together, these data provide strong supporting evidence that the cloned gene cluster identified in this study is responsible for TMC biosynthesis in Streptomyces sp. CK4412, and set the stage for detailed genetic and biochemical studies of the biosynthesis of this important metabolite.

It had been assumed that production of the cytotoxie polyketide mycolactone was strictly associated with Mycobacterium ulcerans, the causative agent of Buruli ulcer. However, a recent study has uncovered a broader distribution of mycolactone-producing mycobacteria (MPM) that includes mycobacteria cultured from diseased fish and frogs in the United States and from diseased fish in the Red and Mediterranean Seas. All of these mycobacteria contain versions of the M. ulcerans pMUM plasmid, produce mycolactones, and show a high degree of genetic relatedness to both M. ulcerans and Mycobacterium marinum. Here, we show by multiple genetic methods, including multilocus sequence analysis and DNA-DNA hybridization, that all MPM have evolved from a common M. marinum progenitor to form a genetically cohesive group among a more diverse assemblage of M. marinum strains. Like M. ulcerans, the fish and frog MPM show multiple copies of the insertion sequence IS2404. Comparisons of pMUM and chromosomal gene sequences demonstrate that plasmid acquisition and the subsequent ability to produce mycolactone were probably the key drivers of speciation. Ongoing evolution among MPM has since produced at least two genetically distinct ecotypes that can be broadly divided into those typically causing disease in ectotherms (but also having a high zoonotic potential) and those causing disease in endotherms, such as humans.

The [approximately equal to] 80-kb pksX gene cluster in Bacillus subtilis encodes an unusual hybrid polyketide/nonribosomal peptide synthase that has been linked to the production of the uncharacterized antibiotic bacillaene. Multiple copies of this synthase, each similar in size to the ribosome, assemble into a single organelle-like complex with a mass of tens to hundreds of megadaltons. The resource requirements of the assembled megacomplex suggest that bacillaene has an important biological role. By coupling a differential NMR spectroscopic technique with genetically manipulated strains of B. subtilis, we were able to characterize the structure of this unusual secondary metabolite, which could not be predicted by using bioinformatic analysis. We report that bacillaene is a linear molecule with two amide bonds: the first links an [alpha]-hydroxy carboxylic acid to a [omega]-amino carboxylic acid containing a conjugated hexaene, and the second links the hexaene-containing carboxylic acid to an ([omega]-1) amino carboxylic acid containing a conjugated triene. Knowledge of bacillaene's structure has enabled us to annotate the pksX gene cluster and should facilitate the study of bacillaene's biosynthesis as well as its biological role in B. subtilis. NMR overlay | nonribosomal peptide | polyketide | [beta]-methyl

Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome ([approximately equal to] 2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies [approximately equal to] 2% of the Bacillus subtilis genome, encodes the subunits of [approximately equal to] 2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs. bacillaene | nonribosomal peptide | polyketide | Streptomyces

Meridamycin is a non-immunosuppressant, FKBP-binding macrocyclic polyketide, which has major potential as a neuroprotectant in a range of neurodegenerative disorders including dementia, Parkinson's disease and ischaemic stroke. A biosynthetic cluster predicted to encode biosynthesis of meridamycin was cloned from the prolific secondary-metabolite-producing strain Streptomyces sp. DSM 4137, not previously known to produce this compound, and specific gene deletion was used to confirm the role of this cluster in the biosynthesis of meridamycin. The meridamycin modular polyketide synthase consists of 14 extension modules distributed between three giant multienzyme proteins. The terminal module is flanked by a highly unusual cytochrome P450-like domain. The characterization of the meridamycin biosynthetic locus in this readily manipulated streptomycete species opens the way to the engineering of new, altered meridamycins of potential therapeutic importance. DOI 10.1099/mic.0.29176-0

Biosynthetic origins of the cyclic 13-desmethyl spirolide C were determined by supplementing cultures of the toxigenic dinoflagellate Alexandrium ostenfeldii with stable isotope-labeled precursors and measuring the incorporation patterns by (super 13)NMR spectroscopy. Results reveal that such cyclic toxins are polyketides and support the biosynthetic pathways, used for other polyether dinoflagellate toxins.

The x-ray crystal structure of a 194-kDa fragment from module 5 of the 6-deoxyerythronolide B synthase has been solved at 2.7 [Angstrom] resolution. Each subunit of the homodimeric protein contains a full-length ketosynthase (KS) and acyl transferase (AT) domain as well as three flanking 'linkers.' The linkers are structurally well defined and contribute extensively to intersubunit or interdomain interactions, frequently by means of multiple highly conserved residues. The crystal structure also reveals that the active site residue Cys-199 of the KS domain is separated from the active site residue Ser-642 of the AT domain by [approximately equal to] 80 [Angstrom]. This distance is too large to be covered simply by alternative positioning of a statically anchored, fully extended phosphopantetheine arm of the acyl carrier protein domain from module 5. Thus, substantial domain reorganization appears necessary for the acyl carrier protein to interact successively with both the AT and the KS domains of this prototypical polyketide synthase module. The 2.7-[Angstrom] KS-AT structure is fully consistent with a recently reported lower resolution, 4.5-[Angstrom] model of fatty acid synthase stucture, and emphasizes the close biochemical and structural similarity between polyketide synthase and fatty acid synthase enzymology. modular megasynthase | multienzyme assembly | polyketide synthase

The pksX gene cluster from Bacillus subtilis is predicted to encode the biosynthesis of an as yet uncharacterized hybrid nonribosomal peptide/polyketide secondary metabolite. We used a combination of biochemical and mass spectrometric techniques to assign functional roles to the proteins AcpK, PksC, PksL, PksF, PksG, PksH, and PksI, and we conclude that they act to incorporate an acetate-derived [beta]-methyl branch on an acetoacetyL-S-carrier protein and ultimately generate a [[DELTA].sup.2]-isoprenyL-S-carrier protein. This work highlights the power of mass spectrometry to elucidate the functions of orphan biosynthetic enzymes, and it details a mechanism by which single-carbon [beta]-branches can be inserted into polyketide-like structures. This pathway represents a noncanonical route to the construction of prenyl units and serves as a prototype for the intersection of isoprenoid and polyketide biosynthetic manifolds in other natural product biosynthetic pathways. mass spectrometry | orphan gene cluster | hybrid nonribosomal peptide|polyketide | polyketide methylation

Although bacterial polyketides are of considerable biomedical interest, the molecular biology of polyketide biosynthesis in Bacillus spp., one of the richest bacterial sources of bioactive natural products, remains largely unexplored. Here we assign for the first time complete polyketide synthase (PKS) gene clusters to Bacillus antibiotics. Three giant modular PKS systems of the trans-acyltransferase type were identified in Bacillus amyloliquefaciens FZB 42. One of them, pks1, is an ortholog of the pksX operon with a previously unknown function in the sequenced model strain Bacillus subtilis 168, while the pks2 and pks3 clusters are novel gene clusters. Cassette mutagenesis combined with advanced mass spectrometric techniques such as matrix-assisted laser desorption ionization--time of flight mass spectrometry and liquid chromatography-electrospray ionization mass spectrometry revealed that the pks1 (bae) and pks3 (dif) gene clusters encode the biosynthesis of the polyene antibiotics bacillaene and diflicidin or oxydifficidin, respectively. In addition, B. subtilis OKB105 (pheA [sfp.sup.0]), a transformant of the B. subtilis 168 derivative JH642, was shown to produce bacillaene, demonstrating that the pksX gene cluster directs the synthesis of that polyketide.

Mycobacterium tuberculosis contains >20 enzymes that require activation by transfer of the 4'-phosphopantetheine moiety of CoA onto a conserved serine residue, a posttranslational modification catalyzed by 4'-phosphopantetheinyl transferases (PPTases). The modified proteins are involved in key metabolic processes such as cell envelope biogenesis and the production of virulence factors. We show that two PPTases conserved in all Mycobacterium spp. and in related genera activate two different subsets of proteins and are not functionally redundant. One enzyme, AcpS, activates the two fatty acid synthase systems of mycobacteria, whereas the other PPTase, PptT, acts on type-I polyketide synthases and non-ribosomal peptide synthases, both of which are involved in the biosynthesis of virulence factors. We demonstrate that both PPTases are essential for Mycobacterium smegmatis viability and that PptT is required for the survival of Mycobacterium bovis bacillus Calmette-Guerin. These enzymes are thus central to the biology of mycobacteria and for mycobacterial pathogenesis and represent promising targets for new antituberculosis drugs. fatty acid synthase | lipid metabolism | mycolic acids | polyketide

The genes encoding the entire cluster, epoA, epoB, epoC, epoD, epoE, and epoF, were redesigned and synthesized to allow for expression in Escherichia coli. The design of the synthetic epothilone genes together with Escherichia coli expression provides the ideal platform for both the biochemical investigation of the epothilone polyketide synthase and the generation of novel biosynthetic epothilone analogues.

The synthesis of 6-deoxyerythronolide B, the macrocyclic aglycone of the antibiotic erythromycin by a polyketide synthase (PKS) that has emerged as the prototypical modular megasynthase is described.

Rifamycin B biosynthesis by Amycolatopsis mediterranei S699 involves a number of unusual modification reactions in the formation of the unique polyketide backbone and decoration of the molecule. A number of genes believed to be involved in the tailoring of rifamycin B were investigated and the results confirmed that the formation of the naphthalene ring moiety of rifamycin takes place during the polyketide chain extension and is catalysed by Rif-Orf19, a 3-(3-hydroxyphenyl)propionate hydroxylase-like protein. The cytochrome P450-dependent monooxygenase encoded by rif-orf5 is required for the conversion of the [DELTA]12, 29 olefinic bond in the polyketide backbone of rifamycin W into the ketal moiety of rifamycin B. Furthermore, Rif-Orf3 may be involved in the regulation of rifamycin B production, as its knock-out mutant produced about 40 % more rifamycin B than the wild-type. The work also revealed that many of the genes located in the cluster are not involved in rifamycin biosynthesis, but might be evolutionary remnants carried over from an ancestral lineage.

The use of Red/ET recombination to generate a large number of shuttle plasmids from a diverse library of chimeric domains for directed evolution of the pikromycin polyketide synthase (PKS) is reported. Red/ET recombination technology is adopted for generating new polyketide as it overcame some of the limitations of traditional approaches for PKS engineering.

Genes encoding a type I polyketide synthase (PKS) (PKS12) and a putative laccase (Gip1) that are required for aurofusarin biosynthesis by Gibberella zeae are isolated. Targeted gene deletion and complementation analyses confirmed that both Gip1 and PKS12 are required for aurofusarin production in Gibberella zeae.

Deltalac-acetogenins that are new acetogenin mimics possessing two n-alkyl tails without an alpha,beta-unsaturated gamma-lactone ring are synthesized and it is suggested that their inhibition mechanism may be different from that of common acetogenins. The results obtained demonstrate that deltalac-acetogenins are a novel type of inhibitor at the terminal electron-transfer step pf bovine complex I.

The beta-ketoacyl-acyl carrier protein synthase (KS) domain of the modular 6-deoxyerythronolide B synthase (DEBS) catalyzes the fundamental chain building reaction of polyketide biosynthesis. The rate constants for the individual biochemical steps are determined by a combination of protein acylation and trasthioesterification experiments.

An examination of the megalomicin and erythromycin polyketide synthase (PKSs) is presented in an Escherichia coli strain metabolically engineered to support complex polyketide biosynthesis. It concluded that production of 6-dEb in shake flask fermentations was undetectable by mass spectrometry in the strain expressing the megalomicin (Meg) PKS genes, whereas 31 mg/L 6-dEB was produced by the strain with erythromycin (DEBS) PKS.

Bacterial symbionts have long been suspected to be the true producers of many drug candidates isolated from marine invertebrates. Sponges, the most important marine source of biologically active natural products, have been frequently hypothesized to contain compounds of bacterial origin. This symbiont hypothesis, however, remained unproven because of a general inability to cultivate the suspected producers. However, we have recently identified an uncultured Pseudomonas sp. symbiont as the most likely producer of the defensive antitumor polyketide pederin in Paederus fuscipes beetles by cloning the putative biosynthesis genes. Here we report closely related genes isolated from the highly complex metagenome of the marine sponge Theonella swinhoei, which is the source of the onnamides and theopederins, a group of polyketides that structurally resemble pederin. Sequence features of the isolated genes clearly indicate that it belongs to a prokaryotic genome and should be responsible for the biosynthesis of almost the entire portion of the polyketide structure that is correlated with antitumor activity. Besides providing further proof for the role of the related beetle symbiont-derived genes, these findings raise intriguing ecological and evolutionary questions and have important general implications for the sustainable production of otherwise inaccessible marine drugs by using biotechnological strategies.

The antifungal polyketide soraphen A is produced by the myxobacterium Sorangium cellulosum So ce26. The slow growth, swarming motility and general intransigence of the strain for genetic manipulations make industrial strain development, large-scale fermentation and combinatorial biosynthetic manipulation of the soraphen producer very challenging. To provide a better host for soraphen A production and molecular engineering, the biosynthetic gene cluster for this secondary metabolite was integrated into the chromosome of Streptomyces lividans ZX7. The upstream border of the gene cluster in Sor. cellulosum was defined by disrupting sorC, which is proposed to take part in the biosynthesis of methoxymalonyl-coenzyme A, to yield a Sor. cellulosum strain with abolished soraphen A production. Insertional inactivation of orf2 further upstream of sore had no effect on soraphen A production. The genes sorR, C, D, F and E thus implicated in soraphen biosynthesis were then introduced into an engineered Str. lividans strain that carried the polyketide synthase genes sorA and sorB, and the methyltransferase gene sorM integrated into its chromosome. A benzoate-coenzyme A ligase from Rhodopseudomonas palustris was also included in some constructs. Fermentations with the engineered Str. lividans strains in the presence of benzoate and/or cinnamate yielded soraphen A. Further feeding experiments were used to delineate the biosynthesis of the benzoyl-coenzyme A starter unit of soraphen A in the heterologous host.

The bacteria in the pallial sinuses of Bugula simplex larvae was identified and whether polyketide synthase (PKS) genes and bryostatin-like polyketides were present in the organism was determined. The reports demonstrate a symbiosis in B. simplex that is similar and evolutionarily related to that in B. neritina.

Mycobacterium ulcerans (MU), an emerging human pathogen harbored by aquatic insects, is the causative agent of Buruli ulcer, a devastating skin disease rife throughout Central and West Africa. Mycolactone, an unusual macrolide with cytotoxic and immunosuppressive properties, is responsible for the massive s.c. tissue destruction seen in Buruli ulcer. Here, we show that MU contains a 174-kb plasmid, pMUM001, bearing a cluster of genes encoding giant polyketide synthases (PKSs), and polyketide-modifying enzymes, and demonstrate that these are necessary and sufficient for mycolactone synthesis. This is a previously uncharacterized example of plasmid-mediated virulence in a Mycobacterium, and the emergence of MU as a pathogen most likely reflects the acquisition of pMUM001 by horizontal transfer. The 12-membered core of mycolactone is produced by two giant, modular PKSs, MLSA1 (1.8 MDa) and MLSA2 (0.26 MDa), whereas its side chain is synthesized by MLSB (1.2 MDa), a third modular PKS highly related to MLSA1. There is an extreme level of sequence identity within the different domains of the MLS cluster (>97% amino acid identity), so much so that the 16 ketosynthase domains seem functionally identical. This is a finding of significant consequence for our understanding of polyketide biochemistry. Such detailed knowledge of mycolactone will further the investigation of its mode of action and the development of urgently needed therapeutic strategies to combat Buruli ulcer.

Fungal type I polyketides (PKs) are synthesized by PK synthases (PKSs) and include well known secondary metabolites such as the anticholesterol drug lovastatin and the potent natural carcinogen aflatoxin. Other type I PKs are known to be virulence factors for some plant pathogens and pigments such as melanin. In this study, a phylogenomic approach was used to investigate the origin and diversity of fungal genes encoding putative PKSs that are predicted to synthesize type I PKs. The resulting genealogy, constructed by using the highly conserved PKS ketosynthase (KS) domain, indicated that: (i) Species within subphylum Pezizomycotina (phylum Ascomycota) but not early diverging ascomycetes, like Saccharomyces cerevisiae (Saccharomycotina) or Schizosaccharomyces pombe (Taphrinomycotina), had large numbers (7-25) of PKS genes. (ii) Bacteria and fungi had separate groups of PKS genes; the few exceptions are the likely result of horizontal gene transfer from bacteria to various sublineages of fungi. (iii) The bulk of genes encoding fungal PKSs fell into eight groups. Four groups were predicted to synthesize variously reduced PKs, and four groups were predicted to make unreduced PKs. (iv) Species within different classes of Pezizomycotina shared the same groups of PKS genes. (v) Different fungal genomes shared few putative orthologous PKS genes, even between closely related genomes in the same class or genus. (vi) The discontinuous distributions of orthologous PKSs among fungal species can be explained by gene duplication, divergence, and gene loss; horizontal gene transfer among fungi does not need to be invoked.

A universal PCR method for the rapid amplification of minimal enediyne polyketide synthase (PKS) genes and the application of this methodology to clone remaining prototypical genes from producers of structurally determined enediynes in both family types are presented. A phylogenetic analysis of the new pool of bona fide enediyne PKS genes, consisting of three from 9-membered producers (neocarzinostatin, C1027, and maduropeptin) and three from 10-membered producers (calicheamicin, dynemicin, and esperamicin), reveals a clear genotypic distinction between the two structural families from which to form a predictive model. The results from this study support the postulation that the minimal enediyne PKS helps define the structural divergence of the enediyne core and provides the key tools for generating enediyne hybrid genes/molecular scaffolds; by using the model, a classification is also provided for the unknown enediyne PKS genes previously identified via genome scanning.

Picromycin synthase (PICS), a multifunctional, modular polyketide synthase (PKS) catalyzes the conversion of methylmalonyl-CoA to narbonolide and 10-deoxymethynolide. PICS modules were each expressed in Escherichia coli with a thioesterase domain at the C-terminus to allow release of polyketide products.

Antiparallel helical interactions between intermodular linkers play an important role in chain transfer of a growing polyketide chain. The linker regions are subjected to structural and mutational analysis in order to understand the mechanism of protein-protein recognition.

Results reveal protein-protein interaction between 6-deoxyerythronolide B synthase 1 and pikromycin polyketide synthase. The thioesterase domain of the latter efficiently catalyzes cyclization of polyketide chain intermediates into 6-membered lactone ring products. Data indicate that the sequence and the length of the C-terminal linker region of the enzymes are similar.

Compound 10 (3,4,7,8-tetramethyl-2,5-dithioglycoluril) was converted in two steps to 12a, which contains a malonate and acetate unit attached to N1 and N6, respectively. These two groups are held in proximity in a manner analogous to the loaded ketosynthase domain of polyketide and fatty acid synthases. After cleavage of the methyl ester by pig liver esterase (PLE), a polar intermediate assigned to acid 15 was separated. This material undergoes conversion to acetoacetylglycoluril 18 upon attempted isolation. The overall conversion thus resembles the decarboxylative condensation catalyzed by ketosynthase. Furthermore, isotope labeling and product studies support a mechanism in which decarboxylation of 15 precedes an intramolecular Claisen-like condensation, as it is believed to occur for the ketosynthase enzymic case. The system thus provides a functional chemical model of the key carbon-carbon bond-forming step in fatty acid and polyketide biosynthesis. Key words: biomimetic, decarboxylation, polyketide, esterase, Claisen condensation.

The loading and initial elongation modules of rifamycin synthetase (LM-MI) bimodular protein facilitates the formation of the phenyl ketide 3-hydroxy-2-methyl-3-phenylpropionate. Data suggest that the production of phenyl ketide appears to be dependent on the covalent loading of benzoate onto LM-MI.

The engineered coenzyme B(sub)12-dependent methylmalonyl-CoA mutase--epimerase pathway produces the (2S)-methylmalonyl-CoA extender unit from the succinyl-CoA, instead of prppionyl-CoA as in the propionyl-CoA carboxylase. Isotopic labeling reveals that in the synthesis of polyketide, the starter and extender units are separately derived from exogenous propionate and methylmalonyl-CoA, respectively.

Results reveal that linker interactions and acyl carrier protein-ketosynthase interactions equally contribute to the module 2-module 3 and the module-4-module 5 interfaces in 6-deoxyerythronolide B synthase. However, nonnatural acyl carrier protein domains presented substrates are accepted by module 2 and 6, which is exploited in the engineering of hybrid polyketide synthases.

Type III polyketide synthases (PKS) generate an array of natural products by condensing multiple acetyl units derived from malonyl-CoA to thioester-linked starter molecules covalently bound in the PKS active site. One strategy adopted by Nature for increasing the functional diversity of these biosynthetic enzymes involves modifying polyketide assembly by altering the preference for starter molecules. Chalcone synthase (CHS) is a ubiquitous plant PKS and the first type III PKS described functionally and structurally. Guided by the three-dimensional structure of CHS, Phe-215 and Phe-265, which are situated at the active site entrance, were targeted for site-directed mutagenesis to diversify CHS activity. The resulting mutants were screened against a panel of aliphatic and aromatic CoA-linked starter molecules to evaluate the degree of starter molecule specificity in CHS. Although wild-type CHS accepts a number of natural CoA thioesters, it does not use N-methylanthraniloyl-CoA as a substrate. Substitution of Phe-215 by serine yields a CHS mutant that preferentially accepts this CoA-thioester substrate to generate a novel alkaloid, namely N-methylanthraniloyltriacetic acid lactone. These results demonstrate that a point mutation in CHS dramatically shifts the molecular selectivity of this enzyme. This structure-based approach to metabolic redesign represents an initial step toward tailoring the biosynthetic activity of plant type III PKS.

Research describes cloning and sequencing of a 205 kilobase DNA region containing candicidin gene cluster, from Streptomyces griseus IMRU 3570, involved in the biosynthesis of candicidin. Candicidin is an aromatic polyene antibiotic active against Candida.

Research describes alteration of enzyme specificity of the acyltransferase domain 4 of 6-deoxyerythronolide B synthase by site-directed mutagenesis, which identifies key residues confering the specificity. Results indicate direct involvement of specific amino acids in the acyltransferase specificity and suggests a route for engineering the specificity of modular polyketides.

As the first structural elucidation of a modular polyketide synthase (PKS) domain, the crystal structure of the macrocycle-forming thioesterase (TE) domain from the 6-deoxyerythronolide B synthase (DEBS) was solved by a combination of multiple isomorphous replacement and multiwavelength anomalous dispersion and refined to an R factor of 24.1% to 2.8-[Angstrom] resolution. Its overall tertiary architecture belongs to the [alpha]/[beta]-hydrolase family, with two unusual features unprecedented in this family: a hydrophobic leucine-rich dimer interface and a substrate channel that passes through the entire protein. The active site triad, comprised of Asp-169, His-259, and Ser-142, is located in the middle of the substrate channel, suggesting the passage of the substrate through the protein. Modeling indicates that the active site can accommodate and orient the 6-deoxyerythronolide B precursor uniquely, while at the same time shielding the active site from external water and catalyzing cyclization by macrolactone formation. The geometry and organization of functional groups explain the observed substrate specificity of this TE and offer strategies for engineering macrocycle biosynthesis. Docking of a homology model of the upstream acyl carrier protein (ACP6) against the TE suggests that the 2-fold axis of the TE dimer may also be the axis of symmetry that determines the arrangement of domains in the entire DEBS. Sequence conservation suggests that all TEs from modular polyketide synthases have a similar fold, dimer 2-fold axis, and substrate channel geometry.

Research has been conducted on the diastereomeric stereotetrads. The preparation of these stereotetrads functionalized at both ends of the formyl and carboxymethyl groups is discussed.

The high degree of diastereofacial selectivity, scope of handling of both aldehydes and ester moieties carbon chain length, and easy purification of aldol products are some of the advantages of the anti-aldol reaction in the synthesis of anti-2-alkyl-3-hydroxycarboxylic acid esters using the (-)-N,N-dibenzylnorephedrine derivatives.

Synthesis of Annonaceous acetogenins, comprising asimicin and bullatacin, by naked carbon skeleton strategy, the convergent , and a hybrid approach is described. A library of 64 sterioisomers of the two compounds is constructed using all the above three approaches. Asimicin and bullatacin possess antitumor, antimalarial, immunosuppressive, and pesticidal properties.

The substrate specificity of modules derived from 6-Deoxyertythronolide B synthase (DEBS) are analyzed. The observed high degree of similarity in the specificity of the modules was tested. Also observed was a distinct preference by the modules for syn diketides over anti diketides. The results are analyzed from the perspective of an evolutionary and protein engineering

Results point out that the utility and malleability of the vinylogous version of the Mukaiyama aldol and Mannich-type addition reactions with heterocyclic triad building modules for the synthesis of a repertoire of annonaceous acetogenin core units and their nitrogen and sulfur analogues.

The synthetic strategy demonstrates the use of chiral allenylindium reagents to access anti,anti and anti,syn stereotriads via additions to beta-oxygenated alpha-methyl aldehydes with C-C bond formation and selective creation of stereocenters and a terminal alkyne. The C5-C20 and C21-C34 subunits of the polyketide macrolides possess 15 stereocenters of the core structure.