The biosynthesis of terpenoids in Gibberella fujikuroi

Abstract

The aim of this work was to study the terpenoid biosynthesis and its regulation in the fungus Gibberella fujikuroi. This was facilitated by the use of a number of mutants of G. fujikuroi produced using UV radiation or N-methyl-N'-nitrosoguanidine and selected mainly by mutation in colour and photoregulation (Avalos et al., 1985). A separation procedure for the carotenoids was devised and up to 9 carotenoids were isolated. Their levels were measured in wild type G. fujikuroi and 4- of the mutants in both light and dark grown conditions. From this a carotenogenic pathway for G. fujikuroi was proposed and the carotenoid levels were shown to be photoregulated. These levels were also affected by the nature of the growth medium used. A method for the separation of 5 of the gibberellins (GA3, GA4, GA7, GA13 and GA-14) was developed using a reverse phase, Spherisorb S50DS HPLC column. This methodology was used to determine time courses for gibberellin production in the wild type and 2 mutant strains. The results showed that the rate of GA production was increased in the mutants, compared to the wild type. Also light and dark grown gibberellin levels were measured in 6 mutants and 2 wild type strains, along with the effects of different media. It was shown, as in carotenogenesis, gibberellin levels were photoinduced and that the levels of photoinduction were increased to differing degrees in the mutants compared to the wild types. The overall gibberellin levels were also shown to be affected by the nature of the growth medium. A cell-free system for G. fujikuroi was developed for theincorporation of mevalonic acid into the terpenoids, which gave the possibility of developing systems for studying individual enzymes in terpenoid biosynthesis. A coupled system was developed for assaying phytoene synthetase using the cell-free system of a non-carotenoid producing mutant to produce substrate for phytoene synthetase coupled to extracts of a high-phytoene producing mutant. This assay was then used in attempts to purify phytoene synthetase.