Chapter ten Aspergillus nidulans as a model system to study secondary metabolism

Summary and Future Studies In this review, we have summarized studies illustrating the strides that have been made in understanding secondary metabolism using A. nidulans as a model system. This organism produces many natural products including ST and PN and has been used as a heterologous host to study the biosynthesis of other natural products including lovastatin. Critical advances in our understanding of fungal secondary metabolism include the discovery of ST and PN biosynthetic gene clusters and the discovery of a G-protein/cAMP/protein kinase A mediated growth pathway in A. nidulans regulating secondary metabolism production. This later pathway coordinates both secondary metabolism and asexual development, similar in spirit, but certainly not in mechanism, to the γ-butyrolactone signaling systems that have been found to simultaneously regulate secondary metabolism and morphological differentiation in bacteria.101 The interwoven coregulation of these two processes may be unraveled through our discovery of LaeA, which plays no major role in development (Bok and Keller, unpublished results). The molecular details of LaeA regulation, found only in secondary metabolite-producing fungi, is the subject of ongoing work in our lab. Where else will the future take this unique fungal model system? Another aspect of eukaryotic (fungal or plant) secondary metabolism that differs distinctively from that of bacterial secondary metabolism is the compartmentalization of biosynthetic precursors into various organelles. For example, the final step of PN biosynthesis (catalyzed by IAT) occurs in the peroxisome.102 Thus, naturally occurring PN side chains must be generated in or, like exogenously provided side chains, be transported into this organelle. The amino acid substrates of PN biosynthesis are sequestered in vacuoles, although ACVS is believed to be cytoplasmic. The synthesis of polyketides, including ST, Draws carbon from the heart of primary metabolism (acetyl-CoA). The acetyl-CoA pool is deliberately divided between the cytoplasm, mitochondria, and peroxisomes to strike the proper balance between energy generation and requisite biosynthetic capabilities (i.e., gluconeogenesis, fatty acid synthesis). How polyketide secondary pathways fit into this network is not yet appreciated. Having only certain subpools of precursor molecules available for secondary metabolic processes could represent an import level of regulation. Knowing which pool of a given metabolite is supplying a secondary pathway could give us insights into how pools are coordinated and could create new opportunities for metabolic engineering. We expect future work on ST and PN biosynthesis in A. nidulans to elaborate more on this important interface between primary and secondary metabolism. The genome sequence of A. nidulans has recently been completed (http://www-genome.wi.mit.edu/annotation/fungi/aspergillus/index.html) and will be a valuable tool for discovery in all aspects of the physiology of this fungus, including sedondary metabolism. Genes required for a given secondary metabolic pathway are invariably clustered in the genome. This is in contrast to other types of genes and likely reflects the importance of horizontal transfer in acquiring these pathways. Thus, genes of secondary metabolic pathways can be predicted just as they are in bacterial genomes. Genes neighboring a PKS- or NRPS-encoding gene are likely required for the same pathway and can be analyzed for coregulation or, if the product of the pathway is known, by deletion analysis. Preliminary BLAST searches of the A. nidulans genome sequence suggest the existence of at least two-dozen polyketide pathways and about a dozen non-ribosomal peptide pathways. Despite this diversity only five of these compounds have been identified: ST, PN, the iron chelator ferricrocin,103 and the polyketides responsible for sexual and asexual spore pigmentation.104,105 A systematic approach could now be taken to delete putative secondary pathway genes and look for alterations in basic physiology and in the production of extractable compounds. The impact of the deletion or overexpression of identified global regulators or individual pathways on the expression of all of the putative secondary pathways could now be assessed with genome-wide transcriptional profiling. More than fifty years after Guido Pontecorvo and coworkers first championed the use of A. nidulans as a genetic model,106 the completed genome sequence has us primed for the next fifty years.