Secondary metabolism in fungi and plants; lipid metabolism and function.
Fungal toxins belong to a class of compounds called secondary metabolites. They have a restricted distribution, sometimes made by only one or a few closely related organisms; they are sometimes made in great quantity but their production varies with growth conditions; and they have no apparent function in the organism producing them. This class of compounds includes not only toxins, but antibiotics, alkaloids, and many other important natural products from plants and fungi.
The fungal toxin patulin, produced by Penicillium patulum and a few related organisms, has become a prototype fungal secondary metabolite because its biosynthetic pathway has been one of the most thoroughly studied. We are interested in studying the regulation of this pathway and the genetic organization of its enzymes. Synthesis of patulin proceeds from the polyketide 6-methylsalicylic acid (6-MS) in a seven-step pathway as outlined below. We have worked out HPLC conditions to assay the production of all the intermediate metabolites of the pathway (including many others formed in side-reactions), and to assay the activity of the enzymes. We are now focusing on isolation and purification of the three enzymes converting m-cresol to phyllostine. These three enzymes are particulate and require oxygen. The first two of the enzymes are hydroxy lases, possibly of the cytochrome P450 type. The third enzyme, an epoxidase, was discovered in our laboratory. Our goal is to obtain either antibodies or sufficient sequence information to construct probes for isolation of the genes of these enzymes. Other laboratories are studying the genetic sequence and organization of other enzymes of the pathway, and we want to complement rather than compete with that effort.
Some earlier work from our laboratory established a variety of conditions for induction of the pathway enzymes. The enzymes are not produced when cells are growing in a balanced way with plenty of nutrients. A point in the culture is reached, referred to as the "tropophase-idiophase transition", at which synthesis of 6-MS is induced, followed shortly by synthesis of the other pathway enzymes. The induction can also be triggered by transfer of the culture from a rich to a poor medium, and by addition of protein synthesis inhibitors. Once we have genetic probes in hand for one or more pathway enzymes, we plan to study the basis for this regulatory behavior.
Our ultimate goal is to understand the genetic basis for the development of complex pathways of secondary metabolism.
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Priest, J.W. and Light, R.J., Patulin Biosynthesis: Epoxidation of Toluquinol and Gentisyl Alcohol by Particulate Preparations from Penicillium patulum. Biochemistry, 28, 9192-9200 (1989).
Priest, J.W. and Light, R.J., Applications of HPLC to Quantitation of Metabolites and Enzymes of the Patulin Pathway from Penicillium patulum. J. Chromatog. 513, 237-246 (1990).
Priest, J.W., and Light, R.J., Patulin Pathway Metabolites from Penicillium patulum. LC Separations. In Atlas of Chromatograms, J. Chromatog. Sci., 29, 273 (1991).