As a means of inducing a reproducible amount of carotenoid synthesis in large quantities of mycelium, a new illumination-incubation technique was developed and is described. In addition, procedures are presented for obtaining phytoene and each of the carotenoid pigments spectroscopically and radiochemically pure when 2-C14_ mevalonate is used as a labelled precursor. The criteria of purity which were employed are discussed.
Using these and other basic procedures, data have been obtained which were all consistent with one particular pathway of carotenoid synthesis. The pathway involves conversion of phytoene to the carotenoid pigments by a series of sequential reactions which include dehydrogenation, cyclization, and oxidative cleavage of a C=C bond.
The proposed pathway is supported by labelling data, time course measurements of each carotenoid in mycelium illuminated and incubated under various physiological conditions, studies with color mutants, and the chemical structures of the compounds involved. In addition, the carotenoid beta-zeacarotene, which had not been previously reported in Neurospora, was shown to be present in mycelium under certain conditions, and this fact added additional support to part of the proposed pathway.
Some information was also obtained about the mechanism of induction of carotenoid synthesis in Neurospora by light. It was confirmed that under well aerated conditions the initial light reaction occurs very rapidly (30 seconds or less). Using cycloheximide, an inhibitor of protein synthesis, evidence was obtained which strongly suggests that one effect of light is to induce the de novo synthesis of an enzyme (or enzymes) which is required for the synthesis of carotenoids and is absent in dark-grown mycelium. Possible mechanisms for such an induction by light are discussed.
