Albert Koulman

Identification of two aflatrem biosynthesis gene loci in Aspergillus flavus and metabolic engineering of Penicillium paxilli to elucidate their function.

ABSTRACT Aflatrem is a potent tremorgenic toxin produced by the soil fungus Aspergillus flavus, and a member of a structurally diverse group of fungal secondary metabolites known as indole-diterpenes. Gene clusters for indole-diterpene biosynthesis have recently been described in several species of filamentous fungi. A search of Aspergillus complete genome sequence data identified putative aflatrem gene clusters in the genomes of A. flavus and Aspergillus oryzae. In both species the genes for aflatrem biosynthesis cluster at two discrete loci; the first, ATM1, is telomere proximal on chromosome 5 and contains a cluster of three genes, atmG, atmC, and atmM, and the second, ATM2, is telomere distal on chromosome 7 and contains five genes, atmD, atmQ, atmB, atmA, and atmP. Reverse transcriptase PCR in A. flavus demonstrated that aflatrem biosynthesis transcript levels increased with the onset of aflatrem production. Transfer of atmP and atmQ into Penicillium paxilli paxP and paxQ deletion mutants, known to accumulate paxilline intermediates paspaline and 13-desoxypaxilline, respectively, showed that AtmP is a functional homolog of PaxP and that AtmQ utilizes 13-desoxypaxilline as a substrate to synthesize aflatrem pathway-specific intermediates, paspalicine and paspalinine. We propose a scheme for aflatrem biosynthesis in A. flavus based on these reconstitution experiments in P. paxilli and identification of putative intermediates in wild-type cultures of A. flavus.

Four gene products are required for the fungal synthesis of the indole‐diterpene, paspaline

Paspaline belongs to a large, structurally and functionally diverse group of indole‐diterpenes synthesized by filamentous fungi. However, the identity of the gene products required for the biosynthesis of paspaline, a key intermediate for the synthesis of paxilline and other indole‐diterpenes, is not known. Transfer of constructs containing different pax gene combinations into a paxilline negative deletion derivative of Penicillium paxilli demonstrated that just four proteins, PaxG, a geranylgeranyl diphosphate synthase, PaxM, a FAD‐dependent monooxygenase, PaxB, a putative membrane protein, and PaxC, a prenyl transferase, are required for the biosynthesis of paspaline.

Defining Paxilline Biosynthesis in Penicillium paxilli FUNCTIONAL CHARACTERIZATION OF TWO CYTOCHROME P450 MONOOXYGENASES

Indole diterpenes are a large, structurally and functionally diverse group of secondary metabolites produced by filamentous fungi. Biosynthetic schemes have been proposed for these metabolites but until recently none of the proposed steps had been validated by biochemical or genetic studies. Using Penicillium paxilli as a model experimental system to study indole diterpene biosynthesis we previously showed by deletion analysis that a cluster of seven genes is required for paxilline biosynthesis. Two of these pax genes, paxP and paxQ (encoding cytochrome P450 monooxygenases), are required in the later steps in this pathway. Here, we describe the function of paxP and paxQ gene products by feeding proposed paxilline intermediates to strains lacking the pax cluster but containing ectopically integrated copies of paxP or paxQ. Transformants containing paxP converted paspaline into 13-desoxypaxilline as the major product and β-PC-M6 as the minor product. β-PC-M6, but not α-PC-M6, was also a substrate for PaxP and was converted to 13-desoxypaxilline. paxQ-containing transformants converted 13-desoxypaxilline into paxilline. These results confirm that paspaline, β-PC-M6, and 13-desoxypaxilline are paxilline intermediates and that paspaline and β-PC-M6 are substrates for PaxP, and 13-desoxypaxilline is a substrate for PaxQ. PaxP and PaxQ also utilized β-paxitriol and α-PC-M6 as substrates converting them to paxilline and α-paxitriol, respectively. These findings have allowed us to delineate clearly the biosynthetic pathway for paxilline for the first time.

Semi-quantitative and structural metabolic phenotyping by direct infusion ion trap mass spectrometry and its application in genetical metabolomics

The identification of quantitative trait loci (QTL) for plant metabolites requires the quantitation of these metabolites across a large range of progeny. We developed a rapid metabolic profiling method using both untargeted and targeted direct infusion tandem mass spectrometry (DIMSMS) with a linear ion trap mass spectrometer yielding sufficient precision and accuracy for the quantification of a large number of metabolites in a high-throughput environment. The untargeted DIMSMS method uses top-down data-dependent fragmentation yielding MS2 and MS3 spectra. We have developed software tools to assess the structural homogeneity of the MS2 and MS3 spectra hence their utility for phenotyping and genetical metabolomics. In addition we used a targeted DIMS(MS) method for rapid quantitation of specific compounds. This method was compared with targeted LC/MS/MS methods for these compounds. The DIMSMS methods showed sufficient precision and accuracy for QTL discovery. We phenotyped 200 individual Lolium perenne genotypes from a mapping population harvested in two consecutive years. Computational and statistical analyses identified 246 nominal m/z bins with sufficient precision and homogeneity for QTL discovery. Comparison of the data for specific metabolites obtained by DIMSMS with the results from targeted LC/MS/MS analysis showed that quantitation by this metabolic profiling method is reasonably accurate. Of the top 100 MS1 bins, 22 ions gave one or more reproducible QTL across the 2 years. Copyright

Analytical methods from the perspective of method standardization

Variability between laboratories, between instruments and between analysts within the same laboratoryis an important issue of practical concern for metabolomics. Method standardization is essentialfor comparability of metabolomics data between experiments and laboratories in multi-disciplinarystudies. However agreed standard requirements to extract metabolites, to concentrate cell extractsand to detect low molecular weight molecules in biological samples are lacking, and this significantlylimits data comparison. This chapter reviews the sources of variation in analytical methods in currentuse and outlines possible quality specifications for global metabolite analysis. We categorize thesources of variability as pre-analytical (sampling and sample preparation), intra-analytical (instrumentation)and post-analytical (data mining and handling). The broad range of applicability of metabolomicsprecludes a generalised uniform approach. However by analyzing the factors influencing metabolitemeasurements, we aim to highlight areas for developing recommendations for method standardizationthat minimize analytical variation and specifications of performance standards including quality controlprocedures and measures of data quality in order to improve laboratory performance and to enable scientistto compare data across studies.

A novel family of cyclic oligopeptides derived from ribosomal peptide synthesis of an in planta-induced gene, gigA, in Epichloë endophytes of grasses.

Fungal endophytes belonging to the genus Epichloë form associations with temperate grasses belonging to the sub-family Poöideae that range from mutualistic through to pathogenic. We previously identified a novel endophyte gene (designated gigA for grass induced gene) that is one of the most abundantly expressed fungal transcripts in endophyte-infected grasses and which is distributed and highly expressed in a wide range of Epichloë grass associations. Molecular and biochemical analyses indicate that gigA encodes a small secreted protein containing an imperfect 27 amino acid repeat that includes a kexin protease cleavage site. Kexin processing of GigA liberates within the plant multiple related products, named here as epichloëcyclins, which we have demonstrated by MS/MS to be cyclic peptidic in nature. Gene deletion of gigA leads to the elimination of all epichloëcyclins with no conspicuous phenotypic impact on the host grass, suggesting a possible bioactive role. This is a further example of a ribosomal peptide synthetic (RiPS) pathway operating within the Ascomycetes, and is the first description of such a pathway from a mutualistic symbiotic fungus from this Phylum.

Deletion and gene expression analyses define the paxilline biosynthetic gene cluster in Penicillium paxilli.

The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were used to define the boundaries of the PAX gene cluster. Targeted gene replacement identified seven genes, paxG, paxA, paxM, paxB, paxC, paxP and paxQ that were all required for paxilline production, with one additional gene, paxD, required for regular prenylation of the indole ring post paxilline synthesis. The two putative transcription factors, PP104 and PP105, were not co-regulated with the pax genes and based on targeted gene replacement, including the double knockout, did not have a role in paxilline production. The relationship of indole dimethylallyl transferases involved in prenylation of indole-diterpenes such as paxilline or lolitrem B, can be found as two disparate clades, not supported by prenylation type (e.g., regular or reverse). This paper provides insight into the P. paxilli indole-diterpene locus and reviews the recent advances identified in paxilline biosynthesis.