Secondary metabolites are low-molecular-weight natural products generated by filamentous fungi, plants, algae, bacteria, and animals in response to environmental abiotic and biotic stimuli. Secondary metabolites have a strong impact on humankind via their application in health, medicine, agriculture, and industry; they include useful (e.g. antibiotics) and detrimental compounds (e.g. mycotoxins).
Filamentous fungi produce a broad range of secondary metabolites. Each fungal species can synthesize multiple secondary metabolites, and these metabolites vary from species to species as well thus enabling the use of secondary metabolite profiling in the chemotaxonomy of filamentous fungi [1–3]. The complex network of secondary metabolism is connected to basic (primary) metabolism. Secondary metabolites are derived from compounds formed during primary metabolism, e.g. amino acids, nucleotides, carbohydrates, acyl-CoA (reviewed in ). Each secondary metabolic pathway accomplishes its specific function (although often unknown) as part of cellular metabolism and appears to provide "active safety" mechanisms for the producer enhancing survival in the continuously changing environment [5–10].
Aspergillus spp. produce an array of secondary metabolites including aflatoxin, cyclopiazonic acid, aflatrem, patulin, penicillin, kojic acid, lovastatin, carotenoids, and spore pigments; novel secondary metabolites have also been discovered that are synthesized from so called silent gene clusters in A. nidulans, such as terrequinone A, monodictyphenone, emodins, and polyketides . Fungal-bacterial physical interactions have been shown to induce silent secondary metabolic gene cluster expression in A. nidulans required for biosynthesis of the polyketide orsellinic acid [5, 9].
Aflatoxin biosynthesis is one of the most highly characterized secondary metabolic pathways [12–15]. In contrast, pathways for the synthesis of many other secondary metabolites, e.g. patulin, cyclopiazonic acid, aflatrem, and kojic acid, are poorly understood [16–18]. Molecular regulation of aflatoxin biosynthesis is complex and involves control of gene expression at the level of the individual gene and at the level of the entire gene cluster [14, 15, 19]. Biosynthesis of aflatoxin initiates during a transition from exponential growth to stationary phase, and closely correlates with fungal development (conidiospore, cleistothecia, and sclerotia formation) [20–22]. AflR, a positive aflatoxin pathway regulator, is a transcription factor that controls at least in part expression of several genes in the aflatoxin gene cluster . VeA, a global regulator of secondary metabolism, links response to light with secondary metabolism and fungal development; this response is mediated through formation of a protein complex VelB/VeA/LaeA . LaeA is a nuclear methyl transferase that through protein-protein interactions mediates regulation of secondary metabolism and development . Aflatoxin biosynthesis is precisely orchestrated within the cell; the early reactions are reported to occur in peroxisomes ; recent evidence from our laboratory suggests that specific early steps as well the middle and late steps are carried out in specialized trafficking vesicles, called aflatoxisomes, which are also involved in export of the toxin outside the cell . A novel role for VeA in coordination of aflatoxisome development with aflatoxin biosynthesis was recently discovered [4, 12]. Biosynthesis of aflatoxins appears to fulfill multiple biochemical and biological functions including removal of acetate, protection of the genome from UV damage , quenching oxidative stress [27–29], protection from insects [30, 31], and regulation of conidiation, and sclerotia development [22, 32–34].
In order to manipulate efficiently secondary metabolism (to enhance production of beneficial metabolites and to control production of detrimental ones) we must understand the "molecular switch" mechanism that controls the initiation of secondary metabolism. Reaching this understanding requires a cooperative effort from genomic, proteomic, and metabolomic research. Despite advances in knowledge about the genes involved in biosynthesis and the regulation of many secondary metabolitic pathways, a detailed understanding of how secondary metabolism integrates with other metabolic and cellular processes is still not available [12, 25, 35–39].
Metabolomics is a powerful tool to characterize the metabolic state of the cell and to discover new metabolites and biochemical pathways . Volatiles, one important group of cellular metabolites, represent a significant portion of the metabolome. Many organic compounds are present in the volatile phase including acids, alcohols, aldehydes, esters, short chain fatty acids, lipid oxides, terpenes, and phenolics. In this study we applied volatile profiling analysis for gaining rapid access to information on intracellular metabolism in the fungus. Specifically, we examined carbon flow in the presence or absence of secondary metabolism in A. parasiticus using wild type and mutant strains carrying genetic defects specifically in aflatoxin biosynthesis and in VeA, a global regulator of secondary metabolism. The volatile metabolites generated by the fungus were analyzed using solid phase microextraction - gas chromatography/mass spectrometry (SPME-GC/MS). This analytical approach is a non-invasive and solvent-free absorption technique that is used in analysis of volatile compounds from the headspace above the sample ; the technique has been widely employed in volatile analysis (profiling) of plants, yeast, and bacteria because it is accurate, sensitive, and robust [41–48]. To conduct this procedure, the outer polymer coating of a fused silica fiber absorbs volatiles from the headspace in the growth environment; the volatiles are then desorbed in the hot GC inlet and chromotographed in the usual manner. The separated compounds are subsequently identified by mass spectrometry.
Using SPME-GC/MS volatile profiling analysis we demonstrated that a genetic block in aflatoxin biosynthesis or disruption of the global regulator veA re-directs intracellular carbon flow. Specifically, we observed that VeA negatively regulates catabolism of branched chain amino acids and the synthesis of ethanol in A. parasiticus; these metabolic changes were mediated at least in part at the transcriptional level. We also showed that volatile metabolites generated under the control of VeA may participate in the molecular machinery that regulates conidiation and sclerotia formation.