Isolation of Bioactive Metabolites from Soil Derived Fungus-Aspergillus fumigatus.

Fungi produce numerous secondary metabolites with intriguing biological properties for the health, industrial, and agricultural sectors. Herein, we report the high-yield isolation of phenolic natural products, N-formyl-4-hydroxyphenyl-acetamide 1 (~117 mg/L) and atraric acid 2 (~18 mg/L), from the ethyl acetate extract of the soil-derived fungus, Aspergillus fumigatus. The structures of compounds 1 and 2 were elucidated through the detailed spectroscopic analysis of NMR and LCMS data. These compounds were assayed for their antimicrobial activities. It was observed that compounds 1 and 2 exhibited strong inhibition against a series of fungal strains but only weak antibacterial properties against multi-drug-resistant strains. More significantly, this is the first known instance of the isolation of atraric acid 2 from a non-lichen fungal strain. We suggest the optimization of this fungal strain may exhibit elevated production of compounds 1 and 2, potentially rendering it a valuable source for the industrial-scale production of these natural antimicrobial compounds. Further investigation is necessary to establish the veracity of this hypothesis.

Unraveling usnic acid: a comparison of biosynthetic gene clusters between two reindeer lichen (Cladonia rangiferina and C. uncialis).

Lichenized fungi are known for their production of a diversity of secondary metabolites, many of which have broad biological and pharmacological applications. By far the most well-studied of these metabolites is usnic acid. While this metabolite has been well-known and researched for decades, the gene cluster responsible for its production was only recently identified from the species Cladonia uncialis. Usnic acid production varies considerably in the genus Cladonia, even among closely related taxa, and many species, such as C. rangiferina, have been inferred to be incapable of producing the metabolite based on analysis by thin-layer chromatography (TLC). We sequenced and examined the usnic acid biosynthetic gene clusters, or lack thereof, from four closely related Cladonia species (C. oricola, C. rangiferina, C. stygia, and C. subtenuis), and compare them against those of C. uncialis. We complement this comparison with tiered chemical profile analyses to confirm the presence or absence of usnic acid in select samples, using both HPLC and LC-MS. Despite long-standing reporting that C. rangiferina lacks the ability to produce usnic acid, we observed functional gene clusters from the species and detected usnic acid when extracts were examined by LC-MS. By contrast, C. stygia and C. oricola, have been previously described as lacking the ability to produce usnic acid, lacked the gene cluster entirely, and no usnic acid could be detected in C. oricola extracts via HPLC or LC-MS. This work suggests that chemical profiles attained through inexpensive and low-sensitivity methods like TLC may fail to detect low abundance metabolites that can be taxonomically informative. This study also bolsters understanding of the usnic acid gene cluster in lichens, revealing differences among domains of the polyketide synthase which may explain observed differences in expression. These results reinforce the need for comprehensive characterization of lichen secondary metabolite profiles with sensitive LC-MS methods.

The isolation, purification and complete characterization of the diterpene forskolin from nutritional supplements.

Forskolin (1) is a diterpene found in the Coleus forskohlii plant that has been examined for its medical properties resulting from adenylyl cyclase activation. This article describes a straightforward purification method of 1 from commercially available weight loss capsules. In addition, there has been some ambiguity with respect to the use of the name 'forskolin' to describe 1 and related diterpenes, which this report serves to eliminate. Herein we detail the complete spectroscopic characterization of purified 1 as well as its single crystal X-ray structure.

Use of cyclic peptides to induce crystallization: case study with prolyl hydroxylase domain 2.

Crystallization is the bottleneck in macromolecular crystallography; even when a protein crystallises, crystal packing often influences ligand-binding and protein-protein interaction interfaces, which are the key points of interest for functional and drug discovery studies. The human hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) readily crystallises as a homotrimer, but with a sterically blocked active site. We explored strategies aimed at altering PHD2 crystal packing by protein modification and molecules that bind at its active site and elsewhere. Following the observation that, despite weak inhibition/binding in solution, succinamic acid derivatives readily enable PHD2 crystallization, we explored methods to induce crystallization without active site binding. Cyclic peptides obtained via mRNA display bind PHD2 tightly away from the active site. They efficiently enable PHD2 crystallization in different forms, both with/without substrates, apparently by promoting oligomerization involving binding to the C-terminal region. Although our work involves a specific case study, together with those of others, the results suggest that mRNA display-derived cyclic peptides may be useful in challenging protein crystallization cases.

Characterization of a Type-2 Diacylglycerol Acyltransferase from Haematococcus pluvialis Reveals Possible Allostery of the Recombinant Enzyme.

Haematococcus pluvialis is a green microalga used in the algal biotechnology industry that can accumulate considerable amounts of storage triacylglycerol (TAG) and astaxanthin, which is a high-value carotenoid with strong antioxidant activity, under stress conditions. Diacylglycerol acyltransferase (DGAT) catalyzes the last step of the acyl-CoA-dependent TAG biosynthesis and appears to represent a bottleneck in algal TAG formation. In this study, putative H. pluvialis DGAT2 cDNA (HpDGAT2A, B, D and E) were identified from a transcriptome database and were subjected to sequence-based in silico analyses. The coding sequences of HpDGAT2B, D, and E were then isolated and characterized through heterologous expression in a TAG-deficient Saccharomyces cerevisiae strain H1246. The expression of HpDGAT2D allowed the recovery of TAG biosynthesis in this yeast mutant, and further in vitro enzymatic assays confirmed that the recombinant HpDGAT2D possessed strong DGAT activity. Interestingly, the recombinant HpDGAT2D displayed sigmoidal kinetics in response to increasing acyl-CoA concentrations, which has not been reported in plant or algal DGAT2 in previous studies.

Phenylacetyl Coenzyme A, Not Phenylacetic Acid, Attenuates CepIR-Regulated Virulence in Burkholderia cenocepacia.

During phenylalanine catabolism, phenylacetic acid (PAA) is converted to phenylacetyl coenzyme A (PAA-CoA) by a ligase, PaaK, and then PAA-CoA is epoxidized by a multicomponent monooxygenase, PaaABCDE, before further degradation through the tricarboxylic acid (TCA) cycle. In the opportunistic pathogen Burkholderia cenocepacia, loss of paaABCDE attenuates virulence factor expression, which is under the control of the LuxIR-like quorum sensing (QS) system, CepIR. To further investigate the link between CepIR-regulated virulence and PAA catabolism, we created knockout mutants of the first step of the pathway (PAA-CoA synthesis by PaaK) and characterized them in comparison to a paaABCDE mutant using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and virulence assays. We found that while loss of PaaABCDE decreased virulence, deletion of the paaK genes resulted in a more virulent phenotype than that of the wild-type strain. Deletion of either paaK or paaABCDE led to higher levels of released PAA but no differences in levels of internal accumulation compared to the wild-type level. While we found no evidence of direct cepIR downregulation by PAA-CoA or PAA, a low-virulence cepR mutant reverted to a virulent phenotype upon removal of the paaK genes. On the other hand, removal of paaABCDE in the cepR mutant did not impact its attenuated phenotype. Together, our results suggest an indirect role for PAA-CoA in suppressing B. cenocepacia CepIR-activated virulence.IMPORTANCE The opportunistic pathogen Burkholderia cenocepacia uses a chemical signal process called quorum sensing (QS) to produce virulence factors. In B. cenocepacia, QS relies on the presence of the transcriptional regulator CepR which, upon binding QS signal molecules, activates virulence. In this work, we found that even in the absence of CepR, B. cenocepacia can elicit a pathogenic response if phenylacetyl-CoA, an intermediate of the phenylacetic acid degradation pathway, is not produced. Instead, accumulation of phenylacetyl-CoA appears to attenuate pathogenicity. Therefore, we have discovered that it is possible to trigger virulence in the absence of CepR, challenging the classical view of activation of virulence by this QS mechanism. Our work provides new insight into the relationship between metabolism and virulence in opportunistic bacteria. We propose that in the event that QS signaling molecules cannot accumulate to trigger a pathogenic response, a metabolic signal can still activate virulence in B. cenocepacia.

Synthesis and antibiotic activity of novel acylated phloroglucinol compounds against methicillin-resistant Staphylococcus aureus.

The rise in antibiotic resistance among pathogenic microorganisms has created an imbalance in the drugs available for treatment, in part due to the slow development of new antibiotics. Cystic fibrosis (CF) patients are highly susceptible to antibiotic-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Phloroglucinols and related polyketide natural products have demonstrated antimicrobial activity against a number of Gram-positive bacteria including S. aureus. In this study, we investigated a series of acylated phloroglucinol derivatives to determine their potential as lead compounds for the design of novel therapeutics. To assess the activity of these compounds, we determined the minimum inhibitory and bactericidal concentration (MIC and MBC, respectively), the minimum biofilm inhibitory and biofilm eradication concentration (MBIC and MBEC, respectively), and evaluated hemolytic activity, as well as their interaction with clinically relevant antibiotics. Of the 12 compounds tested against MRSA and methicillin-susceptible strains, four showed MIC values ranging from 0.125 to 8 µg ml-1 and all of them were bactericidal. However, none of the compounds were able to eradicate biofilms at the concentrations tested. Three of the four did not display hemolytic activity under the conditions tested. Further studies on the interactions of these compounds with clinically relevant antibiotics showed that phlorodipropanophenone displayed synergistic activity when paired with doxycycline. Our results suggest that these acylated phloroglucinols have potential for being further investigated as antibacterial leads.

A comprehensive catalogue of polyketide synthase gene clusters in lichenizing fungi.

Lichens are fungi that form symbiotic partnerships with algae. Although lichens produce diverse polyketides, difficulties in establishing and maintaining lichen cultures have prohibited detailed studies of their biosynthetic pathways. Creative, albeit non-definitive, methods have been developed to assign function to biosynthetic gene clusters in lieu of techniques such as gene knockout and heterologous expressions that are commonly applied to easily cultivatable organisms. We review a total of 81 completely sequenced polyketide synthase (PKS) genes from lichenizing fungi, comprising to our best efforts all complete and reported PKS genes in lichenizing fungi to date. This review provides an overview of the approaches used to locate and sequence PKS genes in lichen genomes, current approaches to assign function to lichen PKS gene clusters, and what polyketides are proposed to be biosynthesized by these PKS. We conclude with remarks on prospects for genomics-based natural products discovery in lichens. We hope that this review will serve as a guide to ongoing research efforts on polyketide biosynthesis in lichenizing fungi.

Lichen ketosynthase domains are not responsible for inoperative polyketide synthases in Ascomycota hosts.

Efforts by lichenologists to characterize lichen polyketide synthases (PKS) through heterologous expression experiments have so far proved unfruitful. A determination of systematic causes of failure is therefore required. Three hypotheses involving the ketosynthase (KS) domain of lichen polyketide synthases (PKS) from Cladonia uncialis are tested: (1) Horizontal versus vertical gene transfer; (2) Typical versus atypical active site residues; (3) Typical versus atypical tertiary protein structure and active site architecture. Phylogenetics, amino acid sequence alignment, and protein modelling indicate that C. uncialis PKS evolved through vertical transfer from Ascomycota fungi, possess Cys-His-His catalytic triads typical of KS from most organisms, and possess protein and catalytic site architecture identical to well-characterized KS from non-lichen organisms. Though the reason for lack of functional activity in heterologous hosts remains unknown, complications involving the KS are ruled out as a likely explanation. Heterologous translation of lichen PKS (or parts thereof) have not been reported. We demonstrate heterologous translation of two lichen KS domains in E. coli.

A c-di-GMP-Modulating Protein Regulates Swimming Motility of Burkholderia cenocepacia in Response to Arginine and Glutamate.

Burkholderia cenocepacia is an opportunistic bacterium that can thrive in different environments, including the amino acid-rich mucus of the cystic fibrosis (CF) lung. B. cenocepacia responds to the nutritional conditions that mimic the CF sputum by increasing flagellin expression and swimming motility. Individual amino acids also induce swimming but not flagellin expression. Here, we show that modulation of the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) levels by the PAS-containing c-di-GMP phosphodiesterase, BCAL1069 (CdpA), regulates the swimming motility of B. cenocepacia K56-2 in response to CF sputum nutritional conditions. Heterologous expression of WspR, a diguanylate cyclase, in B. cenocepacia K56-2 caused an increase in c-di-GMP levels and reduced swimming motility but did not affect flagellin expression or flagellar biosynthesis. After insertional mutagenesis of 12 putative genes encoding c-di-GMP metabolizing enzymes, one mutant of the locus BCAL1069 (cdpA), exhibited decreased swimming motility independent of flagellin expression in CF sputum nutritional conditions and an increase in intracellular c-di-GMP levels. The reduced swimming motility phenotype of the BCAL1069 mutant was observed in the presence of arginine and glutamate, but not of histidine, phenylalanine, or proline. The B. cenocepacia CdpA was also found to be involved in regulation of protease activity but not in biofilm formation. Altogether, these results highlight a role of B. cenocepacia BCAL1069 (CdpA) in sensing the nutritional conditions of the CF sputum and eliciting a pathogenic response that includes swimming motility toward amino acids and an increase in protease activity.

Lichen Biosynthetic Gene Clusters. Part I. Genome Sequencing Reveals a Rich Biosynthetic Potential.

Lichens are symbionts of fungi and algae that produce diverse secondary metabolites with useful properties. Little is known of lichen natural product biosynthesis because of the challenges of working with lichenizing fungi. We describe the first attempt to comprehensively profile the genetic secondary metabolome of a lichenizing fungus. An Illumina platform combined with the Antibiotics and Secondary Metabolites Analysis Shell (FungiSMASH, version 4.0) was used to sequence and annotate assembled contigs of the fungal partner of Cladonia uncialis. Up to 48 putative gene clusters are described comprising type I and type III polyketide synthases (PKS), nonribosomal peptide synthetases (NRPS), hybrid PKS-NRPS, and terpene synthases. The number of gene clusters revealed by this work dwarfs the number of known secondary metabolites from C. uncialis, suggesting that lichenizing fungi have an unexplored biosynthetic potential.

Lichen Biosynthetic Gene Clusters Part II: Homology Mapping Suggests a Functional Diversity.

Lichens are renowned for their diverse natural products though little is known of the genetic programming dictating lichen natural product biosynthesis. We sequenced the genome of Cladonia uncialis and profiled its secondary metabolite biosynthetic gene clusters. Through a homology searching approach, we can now propose specific functions for gene products as well as the biosynthetic pathways that are encoded in several of these gene clusters. This analysis revealed that the lichen genome encodes the required enzymes for patulin and betaenones A-C biosynthesis, fungal toxins not known to be produced by lichens. Within several gene clusters, some (but not all) genes are genetically similar to genes devoted to secondary metabolite biosynthesis in Fungi. These lichen clusters also contain accessory tailoring genes without such genetic similarity, suggesting that the encoded tailoring enzymes perform distinct chemical transformations. We hypothesize that C. uncialis gene clusters have evolved by shuffling components of ancestral fungal clusters to create new series of chemical steps, leading to the production of hitherto undiscovered derivatives of fungal secondary metabolites.

Study of adenylyl cyclase-GαS interactions and identification of novel AC ligands.

Adenylyl cyclases (ACs) are membrane bound enzymes that catalyze the production of cAMP from ATP in response to the activation by G-protein Gαs. Different isoforms of ACs are ubiquitously expressed in different tissues involved in regulatory mechanisms in response to specific stimulants. There are 9 AC isoforms present in humans, with AC5 and AC6 proposed to play a vital role in cardiac functions. The activity of AC6 is sensitive to nitric oxide, such that nitrosylation of the protein might regulate its function. However, the information on structural determinants of nitrosylation in ACs and how they interact with Gαs is limited. Here we used homology modeling to build a molecular model of human AC6 bound to Gαs. Based on this 3D model, we predict the nitrosylation amenable cysteines, and identify potential novel ligands of AC6 using virtual ligand screening. Our model suggests Cys1004 in AC6 (subunit C2) and Cys174 in Gαs present at the AC-Gαs interface as the possible residues that might undergo reversible nitrosylation. Docking analysis predicted novel ligands of AC6 that include forskolin-based compounds and its derivatives. Further work involving site-directed mutagenesis of the predicted residues will allow manipulation of AC activity using novel ligands, and crucial insights on the role of nitrosylation of these proteins in pathophysiological conditions.

Synthetic cystic fibrosis sputum medium diminishes Burkholderia cenocepacia antifungal activity against Aspergillus fumigatus independently of phenylacetic acid production.

Phenylacetic acid (PAA), an intermediate of phenylalanine degradation, is emerging as a signal molecule in microbial interactions with the host. In this work, we explore the presence of phenylalanine and PAA catabolism in 3 microbial pathogens of the cystic fibrosis (CF) lung microbiome: Pseudomonas aeruginosa, Burkholderia cenocepacia, and Aspergillus fumigatus. While in silico analysis of B. cenocepacia J2315 and A. fumigatus Af293 genome sequences showed complete pathways from phenylalanine to PAA, the P. aeruginosa PAO1 genome lacked several coding genes for phenylalanine and PAA catabolic enzymes. High-performance liquid chromatography analysis of supernatants from B. cenocepacia K56-2 detected PAA when grown in Luria-Bertani medium but not in synthetic cystic fibrosis sputum medium (SCFM). However, we were unable to identify PAA production by A. fumigatus or P. aeruginosa in any of the conditions tested. The inhibitory effect of B. cenocepacia on A. fumigatus growth was evaluated using agar plate interaction assays. Inhibition of fungal growth by B. cenocepacia was lessened in SCFM but this effect was not dependent on bacterial production of PAA. In summary, while we demonstrated PAA production by B. cenocepacia, we were not able to link this metabolite with the B. cenocepacia - A. fumigatus microbial interaction in CF nutritional conditions.

Identification of 6-Hydroxymellein Synthase and Accessory Genes in the Lichen Cladonia uncialis.

A transcribed polyketide synthase (PKS) gene has been identified in the lichen Cladonia uncialis. The complete nucleotide sequence of this PKS was determined from the amplified cDNA, and an assignment of individual domains was accomplished by homology searching using AntiSMASH. A scan of the complete genome sequence of C. uncialis revealed the accessory genes associated with this PKS gene. A homology search has identified that several genes in this cluster are similar to genes responsible for the biosynthesis of terrein in Aspergillus terreus. This permitted assignment of putative function to each of the genes in this new C. uncialis cluster. It is proposed that this gene cluster is responsible for the biosynthesis of a halogenated iscoumarin. This is the first report linking a gene cluster to a halogenated metabolite in lichen.

Putative identification of the usnic acid biosynthetic gene cluster by de novo whole-genome sequencing of a lichen-forming fungus.

To identify the biosynthetic gene cluster responsible for the biosynthesis of the polyketide usnic acid we carried out the de novo genome sequencing of the fungal partner of Cladonia uncialis. This was followed by comprehensive in silico annotation of polyketide synthase (PKS) genes. The biosynthesis of usnic acid requires a non-reducing PKS possessing a carbon methylation (CMeT) domain, a terminal Claisen cyclase (CLC) domain, and an accompanying oxidative enzyme that dimerizes methylphloracetophenone to usnic acid. Of the 32 candidate PKS genes identified in the mycobiont genome, only one was identified as consistent with these biosynthetic requirements. This gene cluster contains two genes encoding a non-reducing PKS and a cytochrome p450, which have been respectively named methylphloracetophenone synthase (MPAS) and methylphloracetophenone oxidase (MPAO). Both mpas and mpao were demonstrated to be transcriptionally active by reverse transcriptase-PCR of the mRNA in a lichen sample that was observed by HPLC to produce usnic acid. Phylogenetic analysis of the bioinformatically identified ketosynthase (KS) and CLC domains of MPAS demonstrated that mpas grouped within a unique clade and that mpas could be used as a phylogenetic probe to identify other MPAS genes.

Secondary metabolites from a strain of Alternaria tenuissima Isolated from Northern Manitoba soil.

Deoxyphomalone (1), dimethyl 4-methyl-2,6-pyridinedicarboxylate (2), stemphyperylenol (3), and N-methyl-2-pyrrolidone (4) were isolated from the fermentation broth of a strain of the fungus, Alternaria tenuissima. This fungus was isolated from the soil underlying the lichen, Peltigera didactyla, which had been collected from Wapusk National Park in Northern Manitoba. The structures of the compounds were determined by comprehensive analysis of their spectroscopic data including FT-IR, 1D and 2D NMR spectroscopy and mass spectrometry; and their bioactivities were tested against E.coli cells. The taxonomic identity of the fungus was confirmed by ITS sequencing of its ribosomal DNA.

Limitations of the 'ambush hypothesis' at the single-gene scale: what codon biases are to blame?

Ribosomal frameshifting, a translational error, catastrophically alters the amino acid composition of the nascent protein by shifting the reading frame from the intended contiguous trinucleotide reading. Frameshift events waste energy and resources, and peptide products have unpredictable cytotoxic effects. The 'Ambush Hypothesis' (Seligmann and Pollock 2004, DNA Cell Biol 23:701-5) suggests there is a selective pressure favouring the evolution of out-of-frame ('hidden') stop codons. Although this hypothesis has gained empirical support through whole-genome studies, it is presently unknown whether it can be applied at a single-gene scale. Herein, we report such an investigation using the gene, polyketide synthase (PKS), among species of fungi. Contrary to expectation, genes presented with significantly lower number of hidden stop codons than expected in a selection-neutral model (p < 0.0005), suggesting both non-adherence to the ambush hypothesis as well as suppression of hidden stop codon evolution. It is known that there are multiple adaptive considerations determining codon selection during evolution, and that the information-holding potential of the genetic code is finite. We hypothesize that the reason for low hidden stops in PKS genes is due to competing 'codon biases' that are prioritized over the selective pressure favouring the emergence of hidden stops. Future studies of the ambush hypothesis in the context of other drivers of codon bias may allow this hypothesis to be molded into a comprehensive genetic theory that can be integrated within the broader genetic theory of codon bias and applied to the genetic code at any scale of analysis.

The attenuated virulence of a Burkholderia cenocepacia paaABCDE mutant is due to inhibition of quorum sensing by release of phenylacetic acid.

The phenylacetic acid degradation pathway of Burkholderia cenocepacia is active during cystic fibrosis-like conditions and is necessary for full pathogenicity of B. cenocepacia in nematode and rat infection models; however, the reasons for such requirements are unknown. Here, we show that the attenuated virulence of a phenylacetic acid catabolism mutant is due to quorum sensing inhibition. Unlike wild-type B. cenocepacia, a deletion mutant of the phenylacetyl-CoA monooxygenase complex (ΔpaaABCDE) released phenylacetic acid in the medium that favours infection in Caenorhabditis elegans. Addition of phenylacetic acid further decreased the pathogenicity of the ΔpaaABCDE, which cannot metabolize phenylacetic acid, but did not affect the wild-type, due to phenylacetic acid consumption. In line with reduced detection of acyl-homoserine lactones in spent medium, the ΔpaaABCDE exhibited transcriptional inhibition of the quorum sensing system cepIR. Phenotypes repressed in ΔpaaABCDE, protease activity and pathogenicity against C. elegans, increased with exogenous N-octanoyl-L-homoserine lactone. Thus, we demonstrate that the attenuated phenotype of B. cenocepacia ΔpaaABCDE is due to quorum sensing inhibition by release of phenylacetic acid, affecting N-octanoyl-L-homoserine lactone signalling. Further, we propose that active degradation of phenylacetic acid by B. cenocepacia during growth in cystic fibrosis-like conditions prevents accumulation of a quorum sensing inhibiting compound.

Effect of aposymbiotic conditions on colony growth and secondary metabolite production in the lichen-forming fungus Ramalina dilacerata.

The production of secondary metabolites by aposymbiotic lichen-forming fungi in culture is thought to be influenced by environmental conditions. The effects of the environment may be studied by culturing fungi under defined growing parameters to provide a better understanding of the role of the large number of polyketide synthase (PKS) gene paralogs detected in the genomes of many fungi. The objectives of this study were to examine the effects of culture conditions (media composition and pH level) on the colony growth, the numbers of secondary products, and the expression of two PKS genes by the lichen-forming fungus Ramalina dilacerata. Four types of growth media at four different pH levels were prepared to culture spore isolates of R. dilacerata. Colony diameter and texture were recorded. The number of secondary compounds were determined by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Expression of two PKS genes (non-reducing (NR) and 6-MSAS-type PKS) were compared with expression of an internal control mitochondrial small subunit gene (mtSSU). The results showed that media containing yeast extracts produced the largest colony diameters and the fewest number of secondary metabolites. Colony growth rates also varied with different media conditions, and a significant negative relationship occurred between colony diameter and number of secondary metabolites. Expression of the NR PKS gene was significantly higher at pH 6.5 on the glucose malt agar than any other media, and expression of the 6-MSAS-type (partially-reducing) PKS gene was significantly higher at pH 8.5 on (malt agar) malt agar than on the other types of agar. Gene expression was correlated with the pH level and media conditions that induced the production of the larger number of secondary substances. This is the first study to examine secondary metabolite production in R. dilacerata by comparing the number of polyketides detected with quantitative polymerase chain reaction (qPCR) of two PKS genes under different culture conditions.