Analysis of swainsonine and its early metabolic precursors in cultures of Metarhizium anisopliae

Abstract

The alpha-mannosidase inhibitor swainsonine is produced by the filamentous fungus Metarhizium anisopliae. The primary metabolite pathway from which it is derived is known to be that leading to lysine. In order to effect improvements in the yield of swainsonine it is of interest to study the changes in the intracellular levels of lysine and its biosynthetic intermediates, as well as swainsonine itself, which accompany changes in culture conditions or in the genetics of the microbe. Czapek-Dox defined medium has been used for these studies. A reversed-phase, high performance liquid chromatography procedure was developed for the analysis of lysine, saccharopine, alpha-aminoadipic acid and pipecolic acid in mycelial extracts. The method is based upon precolumn derivatization with 9-fluorenylmethyl chloroformate (FMOC), a reagent known to be useful for the derivatization of amino-containing compounds. Elution with an acetate buffer/acetonitrile gradient effected separation of the four metabolites which were quantified by UV absorption at concentrations from 1 to 20 μg ml-1. Swainsonine concentrations were determined using a previously described enzyme-based method, but applied now to intracellular as well as extracellular samples. Analysis of mycelial extracts from the end of swainsonine accumulation in medium supplemented with L-lysine revealed the accumulation of pipecolic acid and to a lesser extent lysine compared to control mycelium. Controlling the culture medium pH to 9.0 resulted in a drop in swainsonine yield accompanied by an increase in intracellular pipecolic acid levels. Spontaneous mutants tolerant to the presence of the toxic lysine analogue 5-aminoethylcysteine (AEC) were isolated in an attempt to generate lysine over-producers, which might be expected to produce more swainsonine. Surprisingly, four independently isolated mutants produced lower yields of swainsonine, but accumulated higher levels of saccharopine. The tolerance to AEC therefore appears to be due to a reduction in the diversion of saccharopine into swainsonine biosynthesis, allowing the biosynthesis of sufficient lysine to overcome AEC competition.