Katherine M. Williams

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.

The use of yellow fluorescent hybrids to indicate mating in Trypanosoma brucei

BackgroundTrypanosoma brucei undergoes genetic exchange in its insect vector, the tsetse fly, by an unknown mechanism. The difficulties of working with this experimental system of genetic exchange have hampered investigation, particularly because the trypanosome life cycle stages involved cannot be cultured in vitro and therefore must be examined in the insect. Searching for small numbers of hybrid trypanosomes directly in the fly has become possible through the incorporation of fluorescent reporter genes, and we have previously carried out a successful cross using a reporter-repressor strategy. However, we could not be certain that all fluorescent trypanosomes observed in that cross were hybrids, due to mutations of the repressor leading to spontaneous fluorescence, and we have therefore developed an alternative strategy.ResultsTo visualize the production of hybrids in the fly, parental trypanosome clones were transfected with a gene encoding Green Fluorescent Protein (GFP) or Red Fluorescent Protein (RFP). Co-infection of flies with red and green fluorescent parental trypanosomes produced yellow fluorescent hybrids, which were easily visualized in the fly salivary glands. Yellow trypanosomes were not seen in midgut or proventricular samples and first appeared in the glands as epimastigotes as early as 13 days after fly infection. Cloned progeny originating from individual salivary glands had yellow, red, green or no fluorescence and were confirmed as hybrids by microsatellite, molecular karyotype and kinetoplast (mitochondrial) DNA analyses. Hybrid clones showed biparental inheritance of both nuclear and kinetoplast genomes. While segregation and reassortment of the reporter genes and microsatellite alleles were consistent with Mendelian inheritance, flow cytometry measurement of DNA content revealed both diploid and polyploid trypanosomes among the hybrid progeny clones.ConclusionThe strategy of using production of yellow hybrids to indicate mating in trypanosomes provides a robust and unequivocal system for analysis of genetic exchange. Mating occurred with high frequency in these experimental crosses, limited only by the ability of both parental trypanosomes to invade the salivary glands. Yellow hybrids appeared as soon as trypanosomes invaded the salivary glands, implicating the short, unattached epimastigote as the sexual stage. The recovery of diploid, triploid and tetraploid hybrids in these crosses was surprising as genetic markers appeared to have been inherited according to Mendelian rules. As the polyploid hybrids could have been produced from fusion of unreduced gametes, there is no fundamental conflict with a model of genetic exchange involving meiosis.

The programming role of trans-acting enoyl reductases during the biosynthesis of highly reduced fungal polyketides

A novel polyketide synthase nonribosomal peptide synthetase (PKS-NRPS) genecluster was isolated from Beauveria bassiania 992.05. The cluster encodes the enzymes responsible for the biosynthesis of the new 2-pyridone desmethylbassianin (DMB). DMB is structurally related to tenellin from B. bassiana 110.25 but it differs in chain length and degree of methylation. Despite these programming differences the 20 kb DMB biosynthetic genecluster has 90% sequence identity to the tenellingenecluster. Silencing of the PKS-NRPS gene, dmbS, resulted in total loss of DMB production. Co-expression of dmbS in Aspergillus oryzae with its cognate trans-actingenoyl reductase gene, dmbC, produced predesmethylbassianin A, the first isolable precursor in the biosynthetic pathway. Expression of dmbS with the tenellintrans-actingenoyl reductase gene, tenC, also resulted in the production of predesmethylbassianin A. Co-expression of tenS, the tenellin PKS-NRPS, with dmbC produced pretenellin A. These results show that the tenS and dmbS encoded PKS-NRPS contains the programme for polyketide biosynthesis, while the trans-actingERs appear to control the fidelity of the programme. Expression of a hybrid synthetase in which the PKS of the tenellin synthetase was fused to the NRPS from DMBS produced prototenellins A to C, indicating that the NRPS does not act as a selecting gatekeeper to affect the PKS programme.

A toolkit for heterologous expression of metabolic pathways in Aspergillus oryzae.

Much has been learned about the activities of the key enzymes involved in eukaryotic natural product synthesis by isolating the relevant genes and expressing them in a suitable foreign host. Aspergillus oryzae has proved to be an amenable host for the functional analysis of megasynthases from other fungi, but secondary metabolites are often the products of suites of enzymes, and understanding their biosynthesis requires simultaneous expression of several genes. This chapter describes the development and use of a molecular toolkit that facilitates the rapid assembly of the genes constituting whole biosynthetic pathways in one or a few multiple gene expression plasmids designed to provide high-level expression in A. oryzae. Conventional DNA manipulation by restriction/ligation is replaced by homologous recombination in yeast and Gateway®-mediated site-specific recombination in vitro. The toolkit comprises an assembly vector used for the simple construction and modification of large genes from overlapping DNA fragments and three multigene expression vectors. Insertion of three tailoring enzyme genes by homologous recombination and one megasynthase gene by Gateway® transfer into each of the expression vectors can be achieved in a little more than 1 week, and alternative selection markers in the expression plasmids permit cotransformation of A. oryzae with up to 12 genes.

Analysis of a cross between green and red fluorescent trypanosomes

Trypanosoma brucei undergoes genetic exchange in its insect vector, but the mechanism is unknown and no one has yet seen the process. By crossing genetically engineered red and green fluorescent trypanosomes, we have been able to pinpoint the location of genetic exchange in the fly and search for intermediate stages. In experimental crosses of red and green parental trypanosomes, yellow hybrid trypanosomes first appeared in the fly salivary glands as early as 13 days after infection and were observed only in flies with a mixture of red and green trypanosomes in one or both salivary glands. Despite high numbers of flies with mixed infections, yellow trypanosomes were not detected in the fly midgut or proventriculus. The hybrid nature of yellow trypanosomes was confirmed by analysis of molecular karyotypes and microsatellite alleles. As well as yellow hybrids, hybrid trypanosomes with red, green or no fluorescence were also recovered from fly salivary glands. Analysis of microsatellite alleles in parental and progeny clones showed Mendelian inheritance. Our findings are consistent with the hypothesis that mating takes place between trypanosomes in the salivary glands of the fly before they attach to the salivary gland epithelium.

Coomassie blue protein dye-binding assays measure formation of an insoluble protein-dye complex

The solubility of the protein-Coomassie brilliant blue (CBB) complex formed upon Bradford (Anal. Biochem. 72, 248-254, 1976) or Sedmak and Grossberg (Anal. Biochem. 79, 544-552, 1977) protein assay has been investigated by centrifugation or filtration of the assay mix within 10 min of adding dye reagent. The results show complete loss of color yield in the respective supernates and filtrates. This indicates that the protein-CBB complexes are insoluble at the time of absorbance measurement. Protein solubility in the dye reagent may dictate the relative response of the assay to an individual protein and the requirement for macromolecular structure.

Heterologous Production of Fungal Maleidrides Reveals the Cryptic Cyclization Involved in their Biosynthesis

Abstract Fungal maleidrides are an important family of bioactive secondary metabolites that consist of 7, 8, or 9‐membered carbocycles with one or two fused maleic anhydride moieties. The biosynthesis of byssochlamic acid (a nonadride) and agnestadride A (a heptadride) was investigated through gene disruption and heterologous expression experiments. The results reveal that the precursors for cyclization are formed by an iterative highly reducing fungal polyketide synthase supported by a hydrolase, together with two citrate‐processing enzymes. The enigmatic ring formation is catalyzed by two proteins with homology to ketosteroid isomerases, and assisted by two proteins with homology to phosphatidylethanolamine‐binding proteins.

Total Protein Determination in Urine: Aminoglycoside Interference

The aminoglycosides gentamicin and tobramycin interfere in the Dade Behring pyrogallol red-molybdate (PRM) assay used for determination of urinary protein (1). This is an important finding because aminoglycosides are nephrotoxic and accurate determination of urinary protein is necessary to detect renal damage in patients receiving aminoglycosides. We have investigated interference in the PRM assay (2)(3) from other aminoglycosides (dihydrostreptomycin, geneticin, kanamycin, neomycin, paromomycin, and streptomycin) and extended the study to the Coomassie Brilliant Blue (CBB) and the benzethonium chloride (BEC) protein assays, which are also used routinely for urinary protein determination (4)(5)(6). Dihydrostreptomycin (cat. no. D7253), geneticin (cat. no. G5013), gentamicin (cat. no. G3632), kanamycin (cat. no. K4000), neomycin (cat. no. N6386), paromomycin (cat. no. P9297), streptomycin (cat. no. S6501), and tobramycin (cat. no. T1783) were purchased from Sigma-Aldrich Co. Ltd., and solubilized in 0.1 mol/L phosphate buffer (pH …

Biosynthesis of Fungal Polyketides

Fungal polyketides comprise of a large group of structurally diverse compounds with a wide range of biological activities. These compounds are biosynthesised by Type I iterative polyketide synthases (PKS) which can be further defined as non-reducing (nr), partially reducing (pr) and highly reducing (hr) PKS. However, knowledge of the type of PKS is insufficient to predict the actual structure of the final compound. Advances in genome sequencing and bioinformatics technologies, coupled with increased understanding of fungal polyketide tailoring enzymes, make higher-order predictions about the structural family possible. Due to the increased understanding of fungal polyketide biosynthesis and the increased number of pathways characterised, engineering these pathways for the production of novel compounds is becoming more common. This review covers the recent progress in understanding the biosynthesis and engineering of fungal polyketides.