C-methyltransferase Domains Research Articles

C-Methylation controls the biosynthetic programming of alternapyrone.

Alternapyrone is a highly methylated polyene α-pyrone biosynthesised by a highly reducing polyketide synthase. Mutations of the catalytic dyad residues, H1578A/Q and E1604A, of the C-methyltransferase domain resulted in either significantly reduced or no production of alternapyrone, indicating the importance of C-methylation for alternapyrone biosynthesis.

Functional and Structural Analysis of Programmed C-Methylation in the Biosynthesis of the Fungal Polyketide Citrinin

Fungal polyketide synthases (PKSs) are large, multidomain enzymes that biosynthesize a wide range of natural products. A hallmark of these megasynthases is the iterative use of catalytic domains to extend and modify a series of enzyme-bound intermediates. Asubset of these iterative PKSs (iPKSs) contains a C-methyltransferase (CMeT) domain that adds one or more S-adenosylmethionine (SAM)-derived methyl groups to the carbon framework. Neither the basis by which only specific positions on the growing intermediate are methylated ("programming") nor the mechanism of methylation are well understood. Domain dissection and reconstitution of PksCT, the fungal non-reducing PKS (NR-PKS) responsible for the first isolable intermediate in citrinin biosynthesis, demonstrates the role of CMeT-catalyzed methylation in precursor elongation and pentaketide formation. The crystal structure of the S-adenosyl-homocysteine (SAH) coproduct-bound PksCT CMeT domain reveals a two-subdomain organization with a novel N-terminal subdomain characteristic of PKS CMeT domains and provides insights into co-factor and ligand recognition.

The Diversity of Polyketide Synthase Genes from Sugarcane-Derived Fungi

The chemical ecology and biotechnological potential of metabolites from endophytic and rhizosphere fungi are receiving much attention. A collection of 17 sugarcane-derived fungi were identified and assessed by PCR for the presence of polyketide synthase (PKS) genes. The fungi were all various genera of ascomycetes, the genomes of which encoded 36 putative PKS sequences, 26 shared sequence homology with β-ketoacyl synthase domains, while 10 sequences showed homology to known fungal C-methyltransferase domains. A neighbour-joining phylogenetic analysis of the translated sequences could group the domains into previously established chemistry-based clades that represented non-reducing, partially reducing and highly reducing fungal PKSs. We observed that, in many cases, the membership of each clade also reflected the taxonomy of the fungal isolates. The functional assignment of the domains was further confirmed by in silico secondary and tertiary protein structure predictions. This genome mining study reveals, for the first time, the genetic potential of specific taxonomic groups of sugarcane-derived fungi to produce specific types of polyketides. Future work will focus on isolating these compounds with a view to understanding their chemical ecology and likely biotechnological potential.

Mutation of key residues in the C-methyltransferase domain of a fungal highly reducing polyketide synthase

Site directed mutations of the C-methyltransferase domain of squalestatin tetraketide synthase were made in an attempt to alter the methylation pattern of the synthase expressed in vivo: mutation resulted in either no effect or in complete abrogation of polyketide production.

Extensive Mutational Analysis of Modular-Iterative Mixed Polyketide Biosynthesis of Lankacidin inStreptomyces rochei

Extensive mutations of lankacidin synthase genes were carried out to analyze the modular-iterative mixed polyketide biosynthesis of lankacidin. Three ketoreductase domains (lkcC-KR, lkcF-KR1, and lkcF-KR2) were inactivated by in-frame deletion and site-directed mutagenesis of their active sites. The mutants ceased or diminished lankacidin production, indicating that the three KR domains are functional in lankacidin biosynthesis. However, all of the KR mutants failed to accumulate the expected unreduced metabolites. Mutational analysis of two tandemly aligned acyl carrier protein domains (lkcC-ACP1 and lkcC-ACP2) revealed that either ACP is sufficient for lankacidin production. Disruption and complementation experiments on three unique genes/domain (lkcD for acyltransferase, lkcB for dehydratase, and lkcC-MT for a C-methyltransferase domain) suggested that their gene products function iteratively during lankacidin biosynthesis.

Cloning and sequence characterization of a non-reducing polyketide synthase gene from the lichen Xanthoparmelia semiviridis

Lichens produce a diverse array of secondary metabolites that have shown various biological activities. Of particular interest are the coupled phenolics that originate from polyketide pathways, such as depsides, depsidones and usnic acids, which are produced almost solely by lichens. Based on the presumed catalytic domains required for the synthesis of the key intermediates β-orsellinic acid and methylphloroacetophenone, two pairs of degenerate primers were designed to target specifically the β-ketoacylsynthase (KS) and C-methyltransferase (CMeT) domains of fungal non-reducing polyketide synthase (NR-PKS) genes with CMeT domains. These primers were used to explore the genome of the lichen Xanthoparmelia semiviridis, which produces β-orcinol depsidones and usnic acid. One of the two KS domains amplified from genomic DNA of field-collected X. semiviridis was used as a probe to recover the candidate PKS gene. A 13 kb fragment containing an intact putative PKS gene ( xsepks1) of 6555 bp was recovered from a partial genomic library. The inferred amino acid sequence indicated that xsepks1 encodes a protein of 2164 amino acids and contains KS, acyltransferase (AT), acyl carrier protein (ACP) and CMeT domains as expected. This demonstrated a successful strategy for targeting non-reducing PKS genes with CMeT domains. Integration of the 5′ fragment of xsepks1, including the native promoter, into Aspergillus nidulans by cotransformation resulted in the transcription of the 5′ xsepks1 and the splicing of a 63 bp intron, suggesting that A. nidulans could be a suitable heterologous host for xsepks1 expression.

Biosynthesis of the 2‐Pyridone Tenellin in the Insect Pathogenic Fungus Beauveria bassiana

Genomic DNA from the insect pathogenic fungus Beauveria bassiana was used as a template in a PCR with degenerate primers designed to amplify a fragment of a C-methyl transferase (CMeT) domain from a highly reduced fungal polyketide synthase (PKS). The resulting 270-bp PCR product was homologous to other fungal PKS CMeT domains and was used as a probe to isolate a 7.3-kb fragment of genomic DNA from a BamH1 library. Further library probing and TAIL-PCR then gave a 21.9-kb contig that encoded a 12.9-kb fused type I PKS-NRPS ORF together with ORFs encoding other oxidative and reductive enzymes. A directed knockout experiment with a BaR cassette, reported for the first time in B. bassiana, identified the PKS-NRPS as being involved in the biosynthesis of the 2-pyridone tenellin. Other fungal PKS-NRPS genes are known to be involved in the formation of tetramic acids in fungi, and it thus appears likely that related compounds are precursors of 2-pyridones in fungi. B. bassiana tenellin KO and WT strains proved to be equally pathogenic towards insect larvae; this indicated that tenellin is not involved in insect pathogenesis.

Analysis of the Ambruticin and Jerangolid Gene Clusters of Sorangium cellulosum Reveals Unusual Mechanisms of Polyketide Biosynthesis

Ambruticins and jerangolids are structurally related antifungal polyketides produced by Sorangium cellulosum strains. Comparative analysis of the gene clusters and characterization of compounds produced by gene knockout strains suggested hypothetical schemes for biosynthesis of these compounds. Polyketide synthase (PKS) architecture suggests that the pyran ring structure common to ambruticins and jerangolids forms by an intramolecular reaction on a PKS-bound intermediate. Disrupting ambM, encoding a discrete enzyme homologous to PKS C-methyltransferase domains, gave 15-desmethylambruticins. Thus, AmbM is required for C-methylation, but not pyran ring formation. Several steps in the post-PKS modification of ambruticin involve new enzymology. Remarkably, the methylcyclopropane ring and putative carbon atom excision during ambruticin biosynthesis apparently occur on the PKS assembly line. The mechanism probably involves a Favorskii rearrangement, but further work is required to elucidate these complex events.

Polyketide Chain Skipping Mechanism in the Biosynthesis of the Hybrid Nonribosomal Peptide−Polyketide Antitumor Antibiotic Leinamycin in Streptomyces atroolivaceus S-140

A fundamental feature of modular polyketide synthases (PKSs) is the highly predictable relationship between the domain order and the chemical functional groups of resultant polyketide products. Sequence analysis and biochemical characterization of the leinamycin (LNM) biosynthetic gene cluster from Streptomyces atroolivaceus S-140 has revealed a gene, lnmJ, that encodes five PKS modules but with six acyl carrier protein (ACP) domains. The LnmJ PKS module 6 contains two ACP domains, ACP(6-1) and ACP(6-2), separated by a C-methyltransferase domain. Site-directed mutagenesis experiments were carried out with each of these ACPs to test alternative mechanisms proposed for their role in polyketide chain elongation. The in vivo results revealed a new type of polyketide chain "skipping" mechanism, in which either ACP is sufficient for LNM biosynthesis. Biochemical characterization in vitro showed that both ACPs can be loaded with a malonate extender unit by the LnmG acyl transferase; however, ACP(6-2) appears to be preferred because the loading efficiency is about 5-fold that of ACP(6-1). The results are consistent with ACP(6-2) being used for the initial chain elongation step wth ACP(6-1) being involved in the ensuing C-methylation process. These findings provide new insights into the polyketide chain skipping mechanism for modular PKSs.

An Iterative Type I Polyketide Synthase PKSN Catalyzes Synthesis of the Decaketide Alternapyrone with Regio-Specific Octa-Methylation

A biosynthetic gene cluster containing five genes, alt1-5, was cloned from Alternaria solani, a causal fungus of early blight disease to tomato and potato. Homology searching indicated that the alt1, 2, and 3 genes code for cytochrome P450s and the alt4 gene for a FAD-dependent oxygenase/oxidase. The alt5 gene encodes a polyketide synthase (PKS), named PKSN, that was found to be an iterative type I complex reduced-type PKS with a C-methyltransferase domain. To identify the PKSN function, the alt5 gene was introduced into the fungal host Aspergillus oryzae under an alpha-amylase promoter. The transformant produced a polyketide compound, named alternapyrone, whose structure is shown to be 3,5-dimethyl-4-hydroxy-6-(1,3,5,7,11,13-hexamethyl-3,5,11-pentadecatrienyl)-pyran-2-one. Labeling experiments confirmed that alternapyrone is a decaketide with octa-methylation from methionine on every C(2) unit except the third unit.

Fusarin C biosynthesis in Fusarium moniliforme and Fusarium venenatum.

Fragments of polyketide synthase (PKS) genes were amplified from complementary DNA (cDNA) of the fusarin C producing filamentous fungi Fusarium moniliforme and Fusarium venenatum by using degenerate oligonucleotides designed to select for fungal PKS C-methyltransferase (CMeT) domains. The PCR products, which were highly homologous to fragments of known fungal PKS CMeT domains, were used to probe cDNA and genomic DNA (gDNA) libraries of F. moniliforme and F. venenatum. A 4.0 kb cDNA clone from F. venenatum was isolated and used to prepare a hygromycin-resistance knockout cassette, which was used to produce a fusarin-deficient strain of F. venenatum (kb = 1000 bp). Similarly, a 26 kb genomic fragment, isolated on two overlapping clones from F. moniliforme, encoded a complete iterative Type I PKS fused to an unusual nonribosomal peptide synthase module. Once again, targeted gene disruption produced a fusarin-deficient strain, thereby proving that this synthase is responsible for the first steps of fusarin biosynthesis.

Rapid cloning and expression of a fungal polyketide synthase gene involved in squalestatin biosynthesis.

PCR primers designed to selectively amplify the unique C-methyltransferase domain of fungal polyketide synthases were used to selectively clone a polyketide synthase gene involved in the biosynthesis of the squalene synthase inhibitor squalestatin S1 , heterologous expression of which led to the biosynthesis of the squalestatin side-chain.

Design and utility of oligonucleotide gene probes for fungal polyketide synthases

Background: Recent advances in the molecular biology of polyketide biosynthesis have allowed the engineering of polyketide synthases and the biological (‘combinatorial’) synthesis of novel polyketides. Additional structural diversity in these compounds could be expected if more diverse polyketide synthases (PKS) could be utilised. Fungal polyketides are highly variable in structure, reflecting a potentially wide range of differences in the structure and function of fungal PKS complexes. Relatively few fungal synthases have been investigated, perhaps because of a lack of suitable genetic techniques available for the isolation and manipulation of gene clusters from diverse hosts. We set out to devise a general method for the detection of specific PKS genes from fungi. Results: We examined sequence data from known fungal and bacterial polyketide synthases as well as sequence data from bacterial, fungal and vertebrate fatty acid synthases in order to determine regions of high sequence conservation. Using individual domains such as β-ketoacylsynthases (KS), β-ketoreductases (KR) and methyltransferases (MeT) we determined specific short (ca 7 amino acid) sequences showing high conservation for particular functional domains (e.g. fungal KR domains involved in producing partially reduced metabolites; fungal KS domains involved in the production of highly reduced metabolites etc.). Degenerate PCR primers were designed matching these regions of specific homology and the primers were used in PCR reactions with fungal genomic DNA from a number of known polyketide producing species. Products obtained from these reactions were sequenced and shown to be fragments from as-yet undiscovered PKS gene clusters. The fragments could be used in blotting experiments with either homologous or heterologous fungal genomic DNA. Conclusions: A number of sequences are presented which have high utility for the discovery of novel fungal PKS gene clusters. The sequences appear to be specific for particular types of fungal polyketide (i.e. non-reduced, partially reduced or highly reduced KS domains). We have also developed primers suitable for amplifying segments of fungal genes encoding polyketide C-methyltransferase domains. Genomic fragments amplified using these specific primer sequences can be used in blotting experiments and have high potential as aids for the eventual cloning of new fungal PKS gene clusters.