It has been noted since the earliest days of fungal manipulation that many species of filamentous fungi readily synthesize complex compounds that are putatively helpful but not necessary for survival and whose production is presumably costly to maintain. Furthermore, production is often linked to fungal development. Some compounds might function as virulence factors, or their presence could give a competitive edge to the producing organism or enhance the survivability of spores. Some secondary metabolites stimulate sporulation and therefore influence the development of the producing organism and neighboring members of the same species, perhaps enhancing the fitness of a community of related species. Natural products are often produced late in fungal development, and their biosynthesis is complex. This complexity is due to a number of factors that affect secondary metabolite production. These include (i) the influence of a number of external and internal factors on natural product biosynthesis, (ii) the involvement of many sequential enzymatic reactions required for converting primary building blocks into natural products, (iii) tight regulation of natural product enzymatic gene expression by one or more transcriptional activators, (iv) close association of natural product biosynthesis with primary metabolism, and (v) close association of natural products with later stages of fungal development, particularly sporulation. Furthermore, the genes required for biosynthesis of some natural products are clustered, perhaps as a consequence of these factors. Gene clusters contain all or most of the genes required for natural product biosynthesis, and logic suggests that their maintenance could only be selected for if the final natural product conferred some advantage to the producing organism. Moreover, natural product biosynthetic gene clusters can be conserved between organisms, for example the sterigmatocystin-aflatoxin biosynthetic gene cluster in several Aspergillus spp.
As discussed in the preceding sections, some natural products directly enhance sporulation of the producing organisms. Relatively few of these compounds have been described. Other secondary metabolites function to protect the spore; for example, melanin is a protectant from UV damage. Most natural products play no obvious roles in sporulation or spore protection but are secreted into the environment at a time in the life cycle of the fungus that corresponds with sporulation. However, some of the compounds that are excreted into the environment could have subtle effects on the organism that are not immediately obvious. For example, although sterigmatocystin was long thought to have no effect on the producing fungus, recent studies (97
) have demonstrated an effect of sterigmatocystin on Aspergillus
spp. produce mycotoxins at a time that coincides with spore development. In A. nidulans
, these processes are regulated by G-protein signaling pathway components (55
). Inactivation of a G α-subunit, FadA, leads to concomitant sporulation and mycotoxin production. Some of the G-protein signaling regulatory elements described for A. nidulans
are conserved among other filamentous fungi (6
). However, they can have different or even opposite roles in regulating the biosynthesis of natural products (115
). As Tag and collaborators reported, the fadAG42R
allele negatively regulates sterigmatocystin production but positively regulates trichothecene and penicillin production (115
). Why should the same G-protein signaling pathway oppositely regulate two or more different natural products? The particular example of opposite regulation of sterigmatocystin and penicillin production in A. nidulans
by the same signaling pathway allows us to speculate on the necessity of differential regulation when we consider that these two metabolites are also oppositely regulated by pH (penicillin is favored in alkali environments, and sterigmatocystin is favored in acidic environments). Although penicillin is destroyed at low pHa, aflatoxin and sterigmatocystin are very stable molecules not affected by pH extremes. All three of these molecules are complex and incur a large energy cost. Aflatoxin is known to be toxic to insects (90
), while penicillin is a renowned bactericidal antibiotic. One could speculate that in their natural environment, Aspergillus
spp. face more competition from bacteria in alkaline soils, while acidic soils are generally less populous in bacteria. Therefore, penicillin is produced during fungal development in alkaline environments to destroy its bacterial competition, while sterigmatocystin and aflatoxin secretion and accumulation in mycelial and sclerotial tissue in acidic environments provide protection against insects. Penicillin and sterigmatocystin biosynthesis is temporal in nature, with penicillin biosynthetic gene transcription occurring earlier than stc
gene transcription (115
). This would ensure that penicillin is secreted into the environment first to kill off fast-growing prokaryotes, allowing the fungus to establish itself in the community. Later, penicillin production ceases and sterigmatocystin is produced to protect against eukaryotic competitors.
The G-protein signaling pathway operates in most organisms and appears to be a conserved mechanism linking external stimuli to a coordinated response by the organism. In Aspergillus
, homologs of familiar proteins identified in other organisms, such as an RGS domain protein, α- and β-subunits of heterotrimeric G proteins, and cAMP-dependent PKA, are involved in controlling genes involved in conidiation (via brlA
expression) and mycotoxin production (via aflR
expression). Although most of the work on the G-protein signaling pathway linking mycotoxin production and sporulation has been done using Aspergillus
species, particularly A. nidulans
, evidence indicates the presence of a similar pathway regulating tricothecene production in Fusarium
). The G-protein signaling pathway is an appealing mechanism to link fungal development with natural product metabolism for the following reasons: environmental influences on sporulation and sterigmatocystin and aflatoxin production in Aspergillus
spp. can be very complex and contradictory, particularly with regards to pH. If multiple signals for development and mycotoxin production are received external to the cell by different receptors that sense pH, sugar, and nitrogen content of the environment, a system is required to integrate these signals and communicate a coherent message to the nucleus, leading to a coordinated response. Because of the ubiquitous nature of the G-protein signaling pathway in higher organisms, it seems likely that Aspergillus
has coopted this pathway to link sporulation and development with mycotoxin production. We speculate that the same mechanism could operate in other fungi that excrete natural products at the onset of sporulation. However, this signaling pathway might not be the only mechanism to coordinate natural product biosynthesis and sporulation. In liquid shake cultures, Aspergillus
does not sporulate but still produces mycotoxins. Furthermore, some linoleic acid-derived seed compounds can promote sporulation but inhibit aflatoxin gene transcription as described below.
spp. produce mycotoxins during the onset of sporulation. These compounds might influence the fitness of the producing organism but are not considered sporulating factors per se.
However, this genus also secretes linoleic acid-derived molecules, termed psi factors, which do influence asexual and sexual development of the fungus (27
). In addition, some plant unsaturated fatty acids have been shown to influence asexual and sexual development (24
) and mycotoxin production (22
) in this fungus. It is interesting to speculate why Aspergillus
spp. produce sporulating molecules that resemble host seed constituents. Perhaps the production of secondary metabolites that mimic the effect of oilseed compounds on fungal growth and development represents a mechanism for survival by the fungi when it is not feeding on a host seed. For A. nidulans
, a soil organism, the production of linoleic acid-derived psi factors required for development could indicate it evolved from oilseed-infesting aspergilli.
Aflatoxin and sterigmatocystin production in Aspergillus
spp. has been extensively studied due to the deleterious effect of the mycotoxin on human health. While medical investigations have focused on understanding the mechanisms of aflatoxin toxicity and carcinogenicity in humans, these effects of sterigmatocystin and aflatoxin on human health are a consequence of ingesting infected crops and are not the primary function of these compounds. Why these fungi produce sterigmatocystin and aflatoxin; what effect, if any, these compounds have on fungal development; and what ecological role the mycotoxins play are complex questions. In A. nidulans
, mutants that produce no sterigmatocystin or accumulate different intermediates of this mycotoxin have been shown in corn and medium-based experiments to be less fit than wild-type strains (97
), as defined by reduced sporulation. The further one proceeds along the sterigmatocystin pathway, the more fit the organism becomes. Therefore, these results suggest sterigmatocystin can increase the competitive fitness of the organism.
In recent years, great progress has been made in the discovery of signaling pathways that connect fungal development with natural product biosynthesis. However, much remains to be learned. The complexity of these regulatory networks, with multiple target sites and interconnections with other regulatory mechanisms, makes their full elucidation a challenging task. Emerging technologies should be brought to bear on this puzzle. A. nidulans has a large number of mutants available to address these issues. Furthermore, most of the A. nidulans genome has been sequenced and is available (Cereon Inc.). An illuminating set of experiments could involve microarray analysis of genes involved in the G-protein signaling pathway, fatty acid metabolism (as unsaturated fatty acids influence development), acetate metabolism (acetate is derived from the catabolism of fatty acids and is the primary building block for aflatoxin and sterigmatocystin), and sterigmatocystin biosynthesis. Information obtained from the model organism A. nidulans could lead to an understanding of how these regulatory pathways directly or indirectly control fungal development and the biosynthesis of natural products in other microorganisms. This will open up new broad and exciting fields of applications in which the production of beneficial natural products could be enhanced and the production of those with deleterious effects could be reduced or eliminated.