De novo biosynthesis of secondary metabolism enzymes in homogeneous cultures of Penicillium urticae

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Abstract
The initiation of patulin biosynthesis in submerged batch cultures of P. urticae NRRL 2159A was investigated at the enzyme level. In contrast to earlier studies, this study achieved a clear temporal separation of growing cells devoid of secondary metabolism-specific enzymes from nongrowing cells, which rapidly produce these enzymes. A spore inoculum, silicone-treated flasks and 2 new media which supported a rapid, pellet-free, filamentous type of growth were used. In yeast extract-glucose-buffer medium, a marked drop in the specific growth rate (.apprx. 0.26 h-1) coincided with the appearance of the 1st pathway-specific enzyme, 6-methylsalicylic acid synthetase, at about 19 h after inoculation. About 3 h later, when replicatory growth had ceased entirely, the sparsely branched mycelia (length, .apprx. 550 .mu.m) began the rapid synthesis of later pathway enzyme, m-hydroxybenzyl alcohol dehydrogenase. A similar sequence of events occurred in a defined nitrate-glucose-buffer medium; 12 other strains or isolates of P. urticae, and some patulin-producing aspergilli, behaved in a similar manner. The age at which a culture produced m-hydroxybenzyl alcohol dehydrogenase was increased by increasing the nutrient N content of the medium or by decreasing the size of the spore inoculum. In each instance the appearance of enzyme was determined by the nutritional status of the culture and not by its age. A similar appearance of patulin pathway enzymes occurred when a growing culture was resuspended in a N-free 4% glucose solution with or without 0.1 M phosphate (pH 6.5). The appearance of both the synthetase and the dehydrogenase was arrested by the addition of cycloheximide (0.4-5 .mu.g/ml) or actinomycin D (20-80 .mu.g/ml). The requirement for de novo protein and RNA syntheses was confirmed by the incorporation of labeled leucine into the dehydrogenase, and the possibility that latent or preformed proteins were being activated was eliminated.