Anthranilic Acid Accumulation in Saccharomyces cerevisiae Induced by Expression of a Nonribosomal Peptide Synthetase Gene from Paecilomyces cinnamomeus BCC 9616.

Heterologous expression of nrps33, a nonribosomal peptide synthetase gene, from Paecilomyces cinnamomeus BCC 9616 in Saccharomyces cerevisiae unexpectedly resulted in the accumulation of anthranilic acid, an intermediate in tryptophan biosynthesis. Based on transcriptomic and real-time quantitative polymerase chain reaction (RT-qPCR) results, expression of nrps33 affected the transcription of tryptophan biosynthesis genes especially TRP1 which is also the selectable auxotrophic marker for the expression vector used in this work. The product of nrps33 could inhibit the activity of Trp4 involved in the conversion of anthranilate to N-(5'-phosphoribosyl)anthranilate and therefore caused the accumulation of anthranilic acid.

-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 -methyltransferase domain resulted in either significantly reduced or no production of alternapyrone, indicating the importance of -methylation for alternapyrone biosynthesis.

Investigating Fungal Biosynthetic Pathways Using Heterologous Gene Expression: Aspergillus oryzae as a Heterologous Host.

A suite of molecular techniques have been developed in recent decades, which allow gene clusters coding for the biosynthesis of fungal natural products to be investigated and characterized in great detail. Many of these involve the manipulation of the native producer, for example, to increase yields of natural products or investigate the biosynthetic pathway through gene disruptions. However, an alternative and powerful means of investigating biosynthetic pathways, which does not rely on a cooperative native host, is the refactoring and heterologous expression of pathways in a suitable host strain.

Cleaning the Cellular Factory-Deletion of McrA in NSAR1 and the Generation of a Novel Kojic Acid Deficient Strain for Cleaner Heterologous Production of Secondary Metabolites.

The use of filamentous fungi as cellular factories, where natural product pathways can be refactored and expressed in a host strain, continues to aid the field of natural product discovery. Much work has been done to develop host strains which are genetically tractable, and for which there are multiple selectable markers and controllable expression systems. To fully exploit these strains, it is beneficial to understand their natural metabolic capabilities, as such knowledge can rule out host metabolites from analysis of transgenic lines and highlight any potential interplay between endogenous and exogenous pathways.

Fusarochromene, a novel tryptophan-derived metabolite from .

Fusarochromene isolated from the plant pathogenic fungus, Fusarium sacchari is closely related to a group of mycotoxins including fusarochromanone previously isolated from various Fusaria spp. Despite their assumed polyketide biogenesis, incorporation studies with 13C-labelled acetate, glycerol and tryptophans show that fusarochromene is unexpectedly derived via oxidative cleavage of the aromatic amino acid tryptophan. A putative biosynthetic gene cluster has been identified.

SAXS reveals highly flexible interdomain linkers of tandem acyl carrier protein-thioesterase domains from a fungal nonreducing polyketide synthase.

Menisporopsin A is a fungal bioactive macrocyclic polylactone, the biosynthesis of which requires only reducing (R) and nonreducing (NR) polyketide synthases (PKSs) to guide a series of esterification and cyclolactonization reactions. There is no structural information pertaining to these PKSs. Here, we report the solution characterization of singlet and doublet acyl carrier protein (ACP and ACP -ACP )-thioesterase (TE) domains from NR-PKS involved in menisporopsin A biosynthesis.

Assignment of a dubious gene cluster to melanin biosynthesis in the tomato fungal pathogen Cladosporium fulvum.

Pigments and phytotoxins are crucial for the survival and spread of plant pathogenic fungi. The genome of the tomato biotrophic fungal pathogen Cladosporium fulvum contains a predicted gene cluster (CfPKS1, CfPRF1, CfRDT1 and CfTSF1) that is syntenic with the characterized elsinochrome toxin gene cluster in the citrus pathogen Elsinoë fawcettii. However, a previous phylogenetic analysis suggested that CfPks1 might instead be involved in pigment production.

Heterologous biosynthesis of a fungal macrocyclic polylactone requires only two iterative polyketide synthases.

Menisporopsin A is a bioactive macrocyclic polylactone produced by the fungus Menisporopsis theobromae BCC 4162. A scheme for the biosynthesis of this compound has been proposed, in which reducing (R) and non-reducing (NR) polyketide synthases (PKSs) would catalyze the formation of each menisporopsin A subunit, while an additional non-ribosomal peptide synthetase (NRPS)-like enzyme would be required to perform multiple esterification and cyclolactonization reactions. Transcriptome analysis of M.

Identification and manipulation of the pleuromutilin gene cluster from Clitopilus passeckerianus for increased rapid antibiotic production.

Semi-synthetic derivatives of the tricyclic diterpene antibiotic pleuromutilin from the basidiomycete Clitopilus passeckerianus are important in combatting bacterial infections in human and veterinary medicine. These compounds belong to the only new class of antibiotics for human applications, with novel mode of action and lack of cross-resistance, representing a class with great potential. Basidiomycete fungi, being dikaryotic, are not generally amenable to strain improvement.

Production of 3-Oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic Acid in the Fungus Aspergillus oryzae: A Step Towards Heterologous Production of Pyrethrins in Fungi.

Pyrethrins are natural insecticides, which accumulate to high concentrations in pyrethrum (Chrysanthemum cinerariaefolium) flowers. Synthetic pyrethroids are more stable, more efficacious and cheaper, but contemporary requirements for safe and environmentally friendly pesticides encourage a return to the use of natural pyrethrins, and this would be favoured by development of an efficient route to their production by microbial fermentation. The biosynthesis of pyrethrins involves ester linkage between an acid moiety (chrysanthemoyl or pyrethroyl, synthesised via the mevalonic acid pathway from glucose), and an alcohol (pyrethrolone).

Heterologous expression of the avirulence gene from the fungal rice pathogen .

The and genes from the avirulence signalling gene cluster of the rice blast fungus were expressed in and itself. Expression of alone produced a polyenyl pyrone (magnaporthepyrone), which is regioselectively epoxidised and hydrolysed to give different diols, and , in the two host organisms. Analysis of the three introns present in determined that does not process intron 2 correctly, while processes all introns correctly in both appressoria and mycelia.

Production of prostaglandins in transgenic Arabidopsis thaliana.

Plants do not naturally produce the very-long-chain polyunsaturated fatty acids that are the precursors of prostaglandins, but in previous studies Arabidopsis thaliana had been transformed sequentially with genes encoding a Δ(9)-elongase and a Δ(8)-desaturase to produce dihomo-γ-linolenic acid (DGLA) and eicosatetraenoic acid (ETA), and subsequently with a gene encoding a Δ(5)-desaturase to produce arachidonic acid (AA) and eicosapentaenoic acid (EPA). Transformation of A. thaliana with the first two genes consolidated on a single binary vector yielded transformants producing high levels of DGLA, and these plants were further transformed with mouse prostaglandin H synthase (PGH) genes to produce prostaglandins.

Isolation and characterisation of a ferrirhodin synthetase gene from the sugarcane pathogen Fusarium sacchari.

FSN1, a gene isolated from the sugar-cane pathogen Fusarium sacchari, encodes a 4707-residue nonribosomal peptide synthetase consisting of three complete adenylation, thiolation and condensation modules followed by two additional thiolation and condensation domain repeats. This structure is similar to that of ferricrocin synthetase, which makes a siderophore that is involved in intracellular iron storage in other filamentous fungi. Heterologous expression of FSN1 in Aspergillus oryzae resulted in the accumulation of a secreted metabolite that was identified as ferrirhodin.

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.

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.

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.

Nongenetic reprogramming of a fungal highly reducing polyketide synthase.

The biosynthesis of the fungal metabolite tenellin from Beauveria bassiana CBS110.25 was investigated in the presence of the epigenetic modifiers 5-azacytidine and suberoyl bis-hydroxamic acid and under conditions where individual genes from the tenellin biosynthetic gene cluster were silenced. Numerous new compounds were synthesized, indicating that the normal predominant biosynthesis of tenellin is just one outcome out of a diverse array of possible products.

Arachidonic acid: an evolutionarily conserved signaling molecule modulates plant stress signaling networks.

Fatty acid structure affects cellular activities through changes in membrane lipid composition and the generation of a diversity of bioactive derivatives. Eicosapolyenoic acids are released into plants upon infection by oomycete pathogens, suggesting they may elicit plant defenses. We exploited transgenic Arabidopsis thaliana plants (designated EP) producing eicosadienoic, eicosatrienoic, and arachidonic acid (AA), aimed at mimicking pathogen release of these compounds.

Catalytic role of the C-terminal domains of a fungal non-reducing polyketide synthase.

The in vivo activity of truncated forms of methylorcinaldehyde synthase shows that the synthase retains a hydrolytic release activity in the absence of reductive chain release and that chain-length is not controlled by the reductive release domain; experiments using a methyltransferase inhibitor suggest that methylation occurs prior to aromatisation.

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.

Prostanoid production in Saccharomyces cerevisiae provides a novel assay for nonsteroidal anti-inflammatory drugs.

Prostanoids are a large family of lipid mediators originating from prostaglandin H synthase (PGHS) activity on the 20-carbon polyunsaturated fatty acids dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA) and eicosapentaenoic acid. The two mouse PGHS isoforms, PGHS-1 and PGHS-2, were expressed in Saccharomyces cerevisiae (yeast), as was a signal-peptide-deleted version of PGHS-1 (PGHS-1MA). PGHS-1 showed high activity with both AA and DGLA as substrate, whereas PGHS-2 activity was high with DGLA but low with AA.

Auxin transport through non-hair cells sustains root-hair development.

The plant hormone auxin controls root epidermal cell development in a concentration-dependent manner. Root hairs are produced on a subset of epidermal cells as they increase in distance from the root tip. Auxin is required for their initiation and continued growth, but little is known about its distribution in this region of the root.

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.

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).