Some of these metabolites were extracted in relative abundance, but others were present only in minute quantities, such

that it would have been virtually impossible to have isolated them from the initial 200mL extract. Although this example is something of an extreme case, further related metabolites are frequently found from repeat extractions, even those involving no, or a much more modest, scale-up. 3. Maximizing Gene Expression A more general method of generating analogs or related metabolites is to attempt to maximize secondary metabolic gene expression in order to maximize the range of products formed. Although there is no single unified
Fig. 1. Some of the squalestatin group of metabolites.

Follow-Up Natural Product Isolation 467

Fig. 2. Chromatogram of an acidified extract of crude calcium salt of the squalestatins. (A) Typical trace after HPLC of an acidified extract of crude calcium salt. (B) Isolation of crude calcium salt from fermentation of Phoma sp. C2932. (C) Squalestatins isolated in the course of this work and their retention times in a gradient HPLC system. (Reproduced with permission from ref. 4.)

468
Fig. 2. Chromatogram of an acidified extract of crude calcium salt of the squalestatins. (A) Typical trace after HPLC of an acidified extract of crude calcium salt. (B) Isolation of crude calcium salt from fermentation of Phoma sp. C2932. (C) Squalestatins isolated in the course of this work and their retention times in a gradient HPLC system. (Reproduced with permission from ref. 4.)

(A) Typical trace after HPLC of an acidified extract of crude calcium salt. (B) Isolation of crude calcium salt from fermentation of Phoma sp. C2932. (C) Squalestatins isolated in the course of this work and their retention times in a gradient HPLC system. (Reproduced with permission from ref. 4.)

468 Cannell concept of secondary metabolism, it is clear that metabolite production does vary according to an organisms chemical and physical environment. In microbial systems at least, the onset of secondary metabolism, generally coincides with the end of log phase growth and the onset of idiophase, which itself is generally the result of limitation of a specific nutrient. The nature of this limiting nutrient and the other nutrients in the medium can determine the secondary metabolites produced, through processes such as derepression, or inhibition, of secondary metabolic pathways. For instance, carbon catabolite control is exhibited by glucose, which represses phenoxazinone synthase expression and hence actinomycin production in Streptomyces antibioticus; high levels of ammonium suppress the formation of tylosin by Streptomyces fradiae and cephalosporin by Streptomyces clavuligerus; high inorganic phosphate levels repress enzymes involved in the synthesis of tetracyclins, candicidin, neomycin, and streptomycin; concentrations of trace metals (e.g., iron, zinc and cobalt) often have a profound effect on metabolite production (5). Other compounds may induce or enhance the production of secondary metabolites. These include both commonly occurring molecules, such as various amino acids, which may or may not be components of the metabolites, or less prevalent compounds, such as the autoregulator molecules known to play a role in the

related aspects of differentiation and quorum sensing, e.g., A-factor and pamamycin. Overall, the processes that regulate secondary metabolism are poorly understood, a situation that befits a whole form of metabolism whose biological role is still very unclear. Suffice it to say that many physical and chemical parameters can influence secondary metabolite production, and the way in which this can best be used to advantage is generally best determined empirically for any new metabolite or organism. Hence, varying these factors can lead to a much wider range of metabolites than would be produced by an organism under a single set of conditions. This is most easily carried out for microorganisms, though secondary metabolite production by other organisms is also affected by their local environment. Much has been written about this aspect of microbiology (see, for example, ref. 6), and will not be discussed in detail here, but some of the most important factors affecting metabolite production are:
1. Medium: carbon source, nitrogen source, phosphorus source, C/N/P ratios, levels of trace nutrients. 2. Autoregulators (e.g., A-factor and pamamycin).