Zhongshu Song

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.

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.

Authentic Heterologous Expression of the Tenellin Iterative Polyketide Synthase Nonribosomal Peptide Synthetase Requires Coexpression with an Enoyl Reductase

The tenS gene encoding tenellin synthetase (TENS), a 4239‐residue polyketide synthase nonribosomal‐peptide synthetase (PKS‐NRPS) from Beauveria bassiana, was expressed in Aspergillus oryzae M‐2‐3. This led to the production of three new compounds, identified as acyl tetramic acids, and numerous minor metabolites. Consideration of the structures of these compounds indicates that the putative C‐terminal thiolester reductase (R) domain does not act as a reductase, but appears to act as a Dieckmann cyclase (DKC). Expression of tenS in the absence of a trans‐acting ER component encoded by orf3 led to errors in assembly of the polyketide component, giving clues to the mode of programming of highly reducing fungal PKS. Coexpression of tenS with orf3 from the linked gene cluster led to the production of a correctly elaborated polyketide. The NRPS adenylation domain possibly shows the first identified fungal signature sequences for tyrosine selectivity.

Late stage oxidations during the biosynthesis of the 2-pyridone tenellin in the entomopathogenic fungus Beauveria bassiana.

Late stage oxidations during the biosynthesis of the 2-pyridone tenellin in the insect pathogenic fungus Beauveria bassiana were investigated by a combination of gene knockout, antisense RNA, and gene coexpression studies. Open reading frames (ORF) 3 and 4 of the tenellin biosynthetic gene cluster were previously shown to encode a trans-acting enoyl reductase and a hybrid polyketide synthase nonribosomal peptide synthetase (PKS-NRPS), respectively, which together synthesize the acyltetramic acid pretenellin-A. In this work, we have shown that ORF1 encodes a cytochrome P450 oxidase, which catalyzes an unprecedented oxidative ring expansion of pretenellin-A to form the 2-pyridone core of tenellin and related metabolites, and that this enzyme does not catalyze the formation of a hydroxylated precursor. Similar genes appear to be associated with PKS-NRPS genes in other fungi. ORF2 encodes an unusual cytochrome P450 monooxygenase required for the selective N-hydroxylation of the 2-pyridone which is incapable of N-hydroxylation of acyltetramic acids.

Rational Domain Swaps Decipher Programming in Fungal Highly Reducing Polyketide Synthases and Resurrect an Extinct Metabolite

The mechanism of programming of iterative highly reducing polyketide synthases remains one of the key unsolved problems of secondary metabolism. We conducted rational domain swaps between the polyketide synthases encoding the biosynthesis of the closely related compounds tenellin and desmethylbassianin. Expression of the hybrid synthetases in Aspergillus oryzae led to the production of reprogrammed compounds in which the changes to the methylation pattern and chain length could be mapped to the domain swaps. These experiments reveal for the first time the origin of programming in these systems. Domain swaps combined with coexpression of two cytochrome P450 encoding genes from the tenellin biosynthetic gene cluster led to the resurrection of the extinct metabolite bassianin.

Genetic, molecular, and biochemical basis of fungal tropolone biosynthesis

A gene cluster encoding the biosynthesis of the fungal tropolone stipitatic acid was discovered in Talaromyces stipitatus (Penicillium stipitatum) and investigated by targeted gene knockout. A minimum of three genes are required to form the tropolone nucleus: tropA encodes a nonreducing polyketide synthase which releases 3-methylorcinaldehyde; tropB encodes a FAD-dependent monooxygenase which dearomatizes 3-methylorcinaldehyde via hydroxylation at C-3; and tropC encodes a non-heme Fe(II)-dependent dioxygenase which catalyzes the oxidative ring expansion to the tropolone nucleus via hydroxylation of the 3-methyl group. The tropA gene was characterized by heterologous expression in Aspergillus oryzae, whereas tropB and tropC were successfully expressed in Escherichia coli and the purified TropB and TropC proteins converted 3-methylorcinaldehyde to a tropolone in vitro. Finally, knockout of the tropD gene, encoding a cytochrome P450 monooxygenase, indicated its place as the next gene in the pathway, probably responsible for hydroxylation of the 6-methyl group. Comparison of the T. stipitatus tropolone biosynthetic cluster with other known gene clusters allows clarification of important steps during the biosynthesis of other fungal compounds including the xenovulenes, citrinin, sepedonin, sclerotiorin, and asperfuranone.

First Heterologous Reconstruction of a Complete Functional Fungal Biosynthetic Multigene Cluster

Fungal natural products include antibiotics such as the penicillins, antirejection drugs such as cyclosporins and cholesterollowering drugs such as the statins. High productivity of these pharmaceutically important compounds is desirable and has regularly been pursued by strain improvement and metabolic engineering. Recently partial and full genome sequencing has revealed clusters of genes encoding the biosynthesis of these compounds in fungi. Surprisingly it has been found that while rich in bioactive compounds, fungi are even richer in biosynthetic gene clusters. For example, in the well-characterised species Aspergillus nidulans, there are at least 54 secondary metabolite gene clusters, only half of which are characterised at a chemical level. Fungal secondary metabolite gene clusters can be readily identified from key genes within the clusters, such as nonribosomal peptide synthetases (NRPS), terpene cyclases or polyketide synthases (PKS). Although it is relatively easy to identify such gene clusters, it is currently almost impossible to predict the chemical product synthesised, as the programming of the synthase cannot be predicted from sequence information alone. This is further compounded by the presence of genes for many tailoring enzymes, which usually act after the core synthase has completed synthesis of the skeleton, further modifying the resulting compound, making it extremely difficult to elucidate the likely metabolite from gene sequence data alone. A number of different strategies have been employed to decipher fungal gene clusters. These include: chemical profiling of cultures produced under a range of different conditions; over-expression of pathway regulatory genes (where present) ; manipulation of transcriptional activators (e.g. , LaeA) ; manipulation of the pH regulatory system (e.g. , PacC) ; use of chromatin modifying (i.e. , epigenetic) chemicals ; and cofermentation of bacteria and fungi. Unfortunately all of these techniques are restricted in their general utility. Many of the biosynthetic clusters do not include specific transcriptional regulators, many pathways do not respond to LaeA or PacC regulation and chromatin modifiers only activate a small subset of the gene clusters within a genome. In addition, many fungi are difficult to cultivate on a large scale and are often recalcitrant to molecular techniques, so these approaches cannot be applied. We and others have used another approach to investigate the biosynthesis of biologically active compounds in fungi that of heterologous gene expression. This method involves transfer of a gene of interest from a donor strain to a suitable host. In principle a bacterial host such as E. coli could be used, but several problems are usually encountered. First, bacterial hosts are unable to process eukaryotic introns and so these must be removed. Second, expression of eukaryotic genes in bacteria can be problematic, especially if there is a significant codon bias. Third, bacteria can experience difficulty in correctly folding fungal polypeptides. Fourth, proteins such as PKS and NRPS require selective post-translational phosphopantetheinylation for them to be active in vivo. Finally, bacteria may not supply specific metabolites for biosynthesis. While each of these problems can be overcome in isolation, cumulative effects can make the use of bacteria as expression hosts for fungal genes troublesome and inefficient. This is illustrated by the recent expression of the beauvericin NRPS (bbBeas) from

A Natural Plasmid Uniquely Encodes Two Biosynthetic Pathways Creating a Potent Anti-MRSA Antibiotic

Background Understanding how complex antibiotics are synthesised by their producer bacteria is essential for creation of new families of bioactive compounds. Thiomarinols, produced by marine bacteria belonging to the genus Pseudoalteromonas, are hybrids of two independently active species: the pseudomonic acid mixture, mupirocin, which is used clinically against MRSA, and the pyrrothine core of holomycin. Methodology/Principal Findings High throughput DNA sequencing of the complete genome of the producer bacterium revealed a novel 97 kb plasmid, pTML1, consisting almost entirely of two distinct gene clusters. Targeted gene knockouts confirmed the role of these clusters in biosynthesis of the two separate components, pseudomonic acid and the pyrrothine, and identified a putative amide synthetase that joins them together. Feeding mupirocin to a mutant unable to make the endogenous pseudomonic acid created a novel hybrid with the pyrrothine via “mutasynthesis” that allows inhibition of mupirocin-resistant isoleucyl-tRNA synthetase, the mupirocin target. A mutant defective in pyrrothine biosynthesis was also able to incorporate alternative amine substrates. Conclusions/Significance Plasmid pTML1 provides a paradigm for combining independent antibiotic biosynthetic pathways or using mutasynthesis to develop a new family of hybrid derivatives that may extend the effective use of mupirocin against MRSA.

Engineered Thiomarinol Antibiotics Active against MRSA Are Generated by Mutagenesis and Mutasynthesis of Pseudoalteromonas SANK73390

The obligate marine bacterium Pseudoalteromonas spp. SANK73390 produces a series of hybrid antibiotics, thiomarinols A–G (Scheme 1), in which a pyrrothine moiety is linked through an amide to close analogues of the clinically significant antibiotic mupirocin (pseudomonic acids, for example, 7–9) produced by Pseudomonas fluorescens. The pyrrothine-containing holomycin (10), N-propionylholothin (11), thiolutin (12), and aureothricin (13) are also antibiotics but the thiomarinols and mupirocin display particularly potent activity against Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) (MIC< 0.01 mgmL ). Pseudomonic acid A (7) was one of the first of an extensive family of antibiotics produced by the “transAT” class of modular polyketide synthases (PKSs). Identification of the thiomarinol (tml) biosythetic gene cluster by full genome sequencing of SANK73390 showed that it is contained on a 97 kb plasmid consisting almost entirely of the thiomarinol biosynthetic genes. These consist of trans-AT PKSs and associated tailoring genes with high homology to the mupirocin (mup) cluster, along with a nonribosomal peptide synthetase (NRPS) linked to a set of tailoring enzymes similar to that recently shown to control holomycin biosynthesis in Streptomyces clavuligerus. In contrast to thiomarinol A (1), the major mupirocin component, pseudomonic acid A (7) has the 9,10-alkene epoxidized which makes it susceptible to intramolecular rearrangements outside a narrow pH range and limits its clinical utility. Mupirocin inhibits isoleucyl-transfer RNA synthetase. The appended pyrrothine moiety in thiomarinol A improves inhibition of this target, but it is yet to be established whether it also imparts an additional mode of antibacterial action.

A conserved motif flags acyl carrier proteins for β-branching in polyketide synthesis.

Type I PKSs often utilise programmed β-branching, via enzymes of an “HMG-CoA synthase (HCS) cassette”, to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in Acyl Carrier Proteins (ACPs) where β-branching is known. Substituting ACPs confirmed a correlation of ACP type with β-branching specificity. While these ACPs often occur in tandem, NMR analysis of tandem β-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modelling and mutagenesis identified ACP Helix III as a probable anchor point of the ACP-HCS complex whose position is determined by the core. Mutating the core affects ACP functionality while ACP-HCS interface substitutions modulate system specificity. Our method for predicting β-carbon branching expands the potential for engineering novel polyketides and lays a basis for determining specificity rules.