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Resorcylic acid lactones and dihydroxyphenylacetic acid lactones represent important pharmacophores with heat shock response and immune system modulatory activities. The biosynthesis of these fungal polyketides involves a pair of collaborating iterative polyketide synthases (iPKSs): a highly reducing iPKS with product that is further elaborated by a nonreducing iPKS (nrPKS) to yield a 1,3-benzenediol moiety bridged by a macrolactone. Biosynthesis of unreduced polyketides requires the sequestration and programmed cyclization of highly reactive poly-β-ketoacyl intermediates to channel these uncommitted, pluripotent substrates to defined subsets of the polyketide structural space. Catalyzed by product template (PT) domains of the fungal nrPKSs and discrete aromatase/cyclase enzymes in bacteria, regiospecific first-ring aldol cyclizations result in characteristically different polyketide folding modes. However, a few fungal polyketides, including the dihydroxyphenylacetic acid lactone dehydrocurvularin, derive from a folding event that is analogous to the bacterial folding mode. The structural basis of such a drastic difference in the way a PT domain acts has not been investigated until now. We report here that the fungal vs. bacterial folding mode difference is portable on creating hybrid enzymes, and we structurally characterize the resulting unnatural products. Using structure-guided active site engineering, we unravel structural contributions to regiospecific aldol condensations and show that reshaping the cyclization chamber of a PT domain by only three selected point mutations is sufficient to reprogram the dehydrocurvularin nrPKS to produce polyketides with a fungal fold. Such rational control of first-ring cyclizations will facilitate efforts to the engineered biosynthesis of novel chemical diversity from natural unreduced polyketides.

10,11-Dehydrocurvularin is a prevalent fungal phytotoxin with heat shock response and immune-modulatory activities. It features a dihydroxyphenylacetic acid lactone polyketide framework with structural similarities to resorcylic acid lactones like radicicol or zearalenone. A genomic locus was identified from the dehydrocurvularin producer strain Aspergillus terreus AH-02-30-F7 to reveal genes encoding a pair of iterative polyketide synthases (A. terreus CURS1 [AtCURS1] and AtCURS2) that are predicted to collaborate in the biosynthesis of 10,11-dehydrocurvularin. Additional genes in this locus encode putative proteins that may be involved in the export of the compound from the cell and in the transcriptional regulation of the cluster. 10,11-Dehydrocurvularin biosynthesis was reconstituted in Saccharomyces cerevisiae by heterologous expression of the polyketide synthases. Bioinformatic analysis of the highly reducing polyketide synthase AtCURS1 and the nonreducing polyketide synthase AtCURS2 highlights crucial biosynthetic programming differences compared to similar synthases involved in resorcylic acid lactone biosynthesis. These differences lead to the synthesis of a predicted tetraketide starter unit that forms part of the 12-membered lactone ring of dehydrocurvularin, as opposed to the penta- or hexaketide starters in the 14-membered rings of resorcylic acid lactones. Tetraketide N-acetylcysteamine thioester analogues of the starter unit were shown to support the biosynthesis of dehydrocurvularin and its analogues, with yeast expressing AtCURS2 alone. Differential programming of the product template domain of the nonreducing polyketide synthase AtCURS2 results in an aldol condensation with a different regiospecificity than that of resorcylic acid lactones, yielding the dihydroxyphenylacetic acid scaffold characterized by an S-type cyclization pattern atypical for fungal polyketides.

Many microbes can be cultured as single-species communities. Often, these colonies are controlled and maintained via the secretion of metabolites. Such metabolites have been an invaluable resource for the discovery of therapeutics (e.g. penicillin, taxol, rapamycin, epothilone). In this article, written for a special issue on imaging mass spectrometry, we show that MALDI-imaging mass spectrometry can be adapted to observe, in a spatial manner, the metabolic exchange patterns of a diverse array of microbes, including thermophilic and mesophilic fungi, cyanobacteria, marine and terrestrial actinobacteria, and pathogenic bacteria. Dependent on media conditions, on average and based on manual analysis, we observed 11.3 molecules associated with each microbial IMS experiment, which was split nearly 50:50 between secreted and colony-associated molecules. The spatial distributions of these metabolic exchange factors are related to the biological and ecological functions of the organisms. This work establishes that MALDI-based IMS can be used as a general tool to study a diverse array of microbes. Furthermore the article forwards the notion of the IMS platform as a window to discover previously unreported molecules by monitoring the metabolic exchange patterns of organisms when grown on agar substrates.

New beauvericins have been synthesized using the nonribosomal peptide synthetase BbBEAS from the entomopathogenic fungus Beauveria bassiana. Chemical diversity was generated by in vitro chemoenzymatic and in vivo whole cell biocatalytic syntheses using either a B. bassiana mutant or an E. coli strain expressing the bbBeas gene.

This review surveys the biological activities and the iterative and recursive biosynthetic mechanisms of fungal cyclooligomer depsipeptides, and their structural diversification by various combinatorial biosynthetic methods.

No abstract given.

The ascomycetous fungus Nectria haematococca, (asexual name Fusarium solani), is a member of a group of >50 species known as the "Fusarium solani species complex". Members of this complex have diverse biological properties including the ability to cause disease on >100 genera of plants and opportunistic infections in humans. The current research analyzed the most extensively studied member of this complex, N. haematococca mating population VI (MPVI). Several genes controlling the ability of individual isolates of this species to colonize specific habitats are located on supernumerary chromosomes. Optical mapping revealed that the sequenced isolate has 17 chromosomes ranging from 530 kb to 6.52 Mb and that the physical size of the genome, 54.43 Mb, and the number of predicted genes, 15,707, are among the largest reported for ascomycetes. Two classes of genes have contributed to gene expansion: specific genes that are not found in other fungi including its closest sequenced relative, Fusarium graminearum; and genes that commonly occur as single copies in other fungi but are present as multiple copies in N. haematococca MPVI. Some of these additional genes appear to have resulted from gene duplication events, while others may have been acquired through horizontal gene transfer. The supernumerary nature of three chromosomes, 14, 15, and 17, was confirmed by their absence in pulsed field gel electrophoresis experiments of some isolates and by demonstrating that these isolates lacked chromosome-specific sequences found on the ends of these chromosomes. These supernumerary chromosomes contain more repeat sequences, are enriched in unique and duplicated genes, and have a lower G+C content in comparison to the other chromosomes. Although the origin(s) of the extra genes and the supernumerary chromosomes is not known, the gene expansion and its large genome size are consistent with this species' diverse range of habitats. Furthermore, the presence of unique genes on supernumerary chromosomes might account for individual isolates having different environmental niches.

Fungal cyclooligomer depsipeptides such as beauvericin, bassianolide, and enniatins display antibiotic, antifungal, insecticidal, broad-spectrum cancer cell antiproliferative, and cell migration inhibitory activities. We have identified a gene encoding a novel enzyme, ketoisovalerate reductase (KIVR), which is the sole provider of D-hydroxyisovalerate (D-Hiv), a common precursor for cyclooligomer depsipeptide biosynthesis in Beauveria bassiana. KIVR and related hypothetical oxidoreductases encoded in fungal genomes are similar to ketopantoate reductases but not to D-hydroxycarboxylate dehydrogenases. We demonstrate that a KIVR knockout B. bassiana strain can be used for the efficient mutasynthesis of unnatural beauvericin congeners. Simultaneous feeding of precursor analogues enabled the combinatorial mutasynthesis of scrambled beauvericins, some assembled entirely from unnatural precursors. The effects of the introduced structural changes on the antiproliferative and cell migration inhibitory activities of these analogues were evaluated.

Fungal polyketides with the resorcylic acid lactone (RAL) scaffold are of interest for growth stimulation, the treatment of cancer, and neurodegenerative diseases. The RAL radicicol is a nanomolar inhibitor of the chaperone Hsp90, whose repression leads to a combinatorial blockade of cancer-causing pathways. Clustered genes for radicicol biosynthesis were identified and functionally characterized from the endophytic fungus Chaetomium chiversii, and compared to recently described RAL biosynthetic gene clusters. Radicicol production is abolished upon targeted inactivation of a putative cluster-specific regulator, or either of the two polyketide synthases that are predicted to collectively synthesize the radicicol polyketide core. Genomic evidence supports the existence of flavin-dependent halogenases in fungi: inactivation of such a putative halogenase from the C. chiversii radicicol locus yields dechloro-radicicol (monocillin I). Inactivation of a cytochrome P450 epoxidase furnishes pochonin D, a deepoxy-dihydro radicicol analog.

Beauvericin, a cyclohexadepsipeptide ionophore from the entomopathogen Beauveria bassiana, shows antibiotic, antifungal, insecticidal, and cancer cell antiproliferative and antihaptotactic (cell motility inhibitory) activity in vitro. The bbBeas gene encoding the BbBEAS nonribosomal peptide synthetase was isolated from B. bassiana and confirmed to be responsible for beauvericin biosynthesis by targeted disruption. BbBEAS utilizes D-2-hydroxyisovalerate (D-Hiv) and L-phenylalanine (Phe) for the iterative synthesis of a predicted N-methyl-dipeptidol intermediate, and forms the cyclic trimeric ester beauvericin from this intermediate in an unusual recursive process. Heterologous expression of the bbBeas gene in Escherichia coli to produce the 3189 amino acid, 351.9 kDa BbBEAS enzyme provided a strain proficient in beauvericin biosynthesis. Comparative infection assays with a BbBEAS knockout B. bassiana strain against three insect hosts revealed that beauvericin plays a highly significant but not indispensable role in virulence.

Precursor-directed biosynthesis was used to produce analogues of the cyclic depsipeptide mycotoxin beauvericin (1) using the filamentous fungus Beauveria bassiana ATCC 7159. Feeding 30 analogues of D-2-hydroxyisovalerate and L-phenylalanine, the natural 2-hydroxycarboxylic acid and amino acid precursors of beauvericin, led to the biosynthesis of novel beauvericins. Six of these were isolated and characterized, and their cytotoxicity and directional cell migration (haptotaxis) inhibitory activity against the metastatic prostate cancer cell line PC-3M were evaluated. Replacement of one, two, or all three of the D-2-hydroxyisovalerate constituents in beauvericin (1) with 2-hydroxybutyrate moieties (beauvericins G(1-3), compounds 2-4) caused a parallel decline of cell migration inhibitory activity and cytotoxicity, suggesting a requirement for a branched side chain for both of these biological activities at the corresponding positions of beauvericins. Replacement of one, two, or all three N-methyl-L-phenylalanine residues of beauvericin with N-methyl-L-3-fluorophenylalanine moieties (beauvericins H(1-3), compounds 5-7) increased cytotoxicity without affecting antihaptotactic activity.

Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4''-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4''-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.

4''-Oxo-avermectin is a key intermediate in the manufacture of the insecticide emamectin benzoate from the natural product avermectin. Seventeen Streptomyces strains with the ability to oxidize avermectin to 4''-oxo-avermectin in a regioselective manner have been discovered, and the enzymes responsible for this reaction were found to be CYPs (cytochrome P450 mono-oxygenases). The genes for these enzymes have been cloned, sequenced and compared to reveal a new subfamily of CYPs. The biocatalytic enzymes have been overexpressed in Escherichia coli, Streptomyces lividans and solvent-tolerant Pseudomonas putida strains using different promoters and vectors. FDs (ferredoxins) and FREs (ferredoxin:NADP(+) reductases) were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains, and tested in co-expression systems to optimize the electron transport. Subsequent studies showed that increasing the biocatalytic conversion levels to commercial relevance results in the production of several side products in significant amounts. Chimaeric Ema CYPs were created by sequential rounds of GeneReassembly, a proprietary directed evolution method, and selected for improved substrate specificity by high-throughput screening.

The cytochrome P450 monooxygenase Ema1 from Streptomyces tubercidicus R-922 and its homologs from closely related Streptomyces strains are able to catalyze the regioselective oxidation of avermectin into 4"-oxo-avermectin, a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate (V. Jungmann, I. Molnár, P. E. Hammer, D. S. Hill, R. Zirkle, T. G. Buckel, D. Buckel, J. M. Ligon, and J. P. Pachlatko, Appl. Environ. Microbiol. 71:6968-6976, 2005). The gene for Ema1 has been expressed in Streptomyces lividans, Streptomyces avermitilis, and solvent-tolerant Pseudomonas putida strains using different promoters and vectors to provide biocatalytically competent cells. Replacing the extremely rare TTA codon with the more frequent CTG codon to encode Leu4 in Ema1 increased the biocatalytic activities of S. lividans strains producing this enzyme. Ferredoxins and ferredoxin reductases were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains and tested in ema1 coexpression systems to optimize the electron transport towards Ema1.

4"-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active Streptomyces strains with the ability to oxidize avermectin to 4"-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in Escherichia coli and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined.

The myxobacterium Sorangium cellulosum So ce26, the producer of the agriculturally important fungicide antibiotic soraphen A, displays coordinated gliding motility (swarming) on agar surfaces. The consequent failure to form detached colonies represents a major obstacle for microbiological and genetic studies, since single cells representing discrete genetic events cannot be reliably separated and propagated as clones. The MglA protein, the product of the mglA gene, has been shown to be a central regulator of gliding motility and swarming in the related myxobacterium Myxococcus xanthus. We have cloned and sequenced a chromosomal locus from S. cellulosum So ce26 that shows similarity to the M. xanthus mglA locus. Insertional inactivation of the chromosomal copy of the S. cellulosum So ce26 mglA homolog resulted in a strain with a non-swarming colony phenotype. This strain is able to form distinct colonies presumably derived from single cells. This is the first report on the characterization of a genetic element of the gliding motility system in the myxobacterial suborder Sorangineae.

A 108-kb genomic DNA region of Saccharopolyspora spinosa NRRL 18395, producer of the agriculturally important insecticidal antibiotics spinosyns, has been cloned, sequenced and analyzed to reveal clustered genes encoding a type I polyketide synthase (PKS) complex. The genes for the PKS are flanked by genes encoding homologs of enzymes that are involved in the urea cycle, valine, leucine and isoleucine biosynthesis and energy metabolism. While the disruption of the PKS genes by insertional inactivation was not expected to abolish the production of spinosyns, no differences were found in the antibacterial, antifungal, or insecticidal activities either of the parental and the knockout mutant strains under the growth conditions tested. Deduction of the most likely structure of the polyketide core of the cryptic metabolite, termed obscurin, from the predicted modules and domains of the PKS suggests the formation of a highly unsaturated substituted C22 carboxylic acid that might undergo further processing after its release from the PKS.

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 sorC 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.

2-Hexyl-5-propylresorcinol is the predominant analog of several dialkylresorcinols produced by Pseudomonas aurantiaca (Pseudomonas fluorescens BL915). We isolated and characterized three biosynthetic genes that encode an acyl carrier protein, a beta-ketoacyl-acyl carrier protein synthase III, and a protein of unknown function, all of which collectively allow heterologous production of 2-hexyl-5-propylresorcinol in Escherichia coli. Two regulatory genes exhibiting similarity to members of the AraC family of transcriptional regulators are also present in the identified gene cluster. Based on the deduced functions of the proteins encoded by the gene cluster and the observed incorporation of labeled carbons from octanoic acid into 2-hexyl-5-propylresorcinol, we propose that dialkylresorcinols are derived from medium-chain-length fatty acids by an unusual head-to-head condensation of beta-ketoacyl thioester intermediates. Genomic evidence suggests that there is a similar pathway for the biosynthesis of the flexirubin-type pigments in certain bacteria belonging to the order Cytophagales.

A genomic DNA region of over 80 kb that contains the complete biosynthetic gene cluster for the synthesis of the antifungal polyketide metabolite soraphen A was cloned from Sorangium cellulosum So ce26. The nucleotide sequence of the soraphen A gene region, including 67,523 bp was determined. Examination of this sequence led to the identification of two adjacent type I polyketide synthase (PKS) genes that encode the soraphen synthase. One of the soraphen A PKS genes includes three biosynthetic modules and the second contains five additional modules for a total of eight. The predicted substrate specificities of the acyltransferase (AT) domains, as well as the reductive loop domains identified within each module, are consistent with expectations from the structure of soraphen A. Genes were identified in the regions flanking the two soraphen synthase genes that are proposed to have roles in the biosynthesis of soraphen A. Downstream of the soraphen PKS genes is an O-methyltransferase (OMT) gene. Upstream of the soraphen PKS genes there is a gene encoding a reductase and a group of genes that are postulated to have roles in the synthesis of methoxymalonyl-acyl carrier protein (ACP). This unusual extender unit is proposed to be incorporated in two positions of the soraphen polyketide chain. One of the genes in this group contains distinct domains for an AT, an ACP, and an OMT.