The biosynthesis of carotenoids is thought to proceed according to the scheme depicted in Fig. 1. From the first 40-carbon compound, phytoene, a series of four dehydrogenations leads to the production of lycopene, two further cyclizations convert it to 0-carotene. j3-Zeacarotene substitutes for lycopene as an intermediate in an alternative proposal, which changes the order of the last dehydrogenation and the first cyclization (1, 2).The discovery of multienzyme systems in the synthesis of fatty acids and other compounds (3-5) has led to speculations (6) that carotenoid synthesis might be organized in an analogous way, but no evidence has yet been found for such a multienzyme aggregate for carotenoids.The wild types of the fungus Phycomyces blakesleeanus accumulate large amounts of ,B-carotene, resulting in bright yellow mycelia, sporangiophores, and young sporangia. cumulating phytoene or unable to synthesize carotenoids altogether) have been isolated (7,8).Phycomyces heterokaryons containing two types of nuclei, one type from a strain failing to produce carotenoids and the other from a strain accumulating lycopene, are of many different shades of color, from whitish to orange, according to the proportion of the two types of nuclei (9). This observation led us to think that perhaps a mixture of carotenoids was resulting from the operation of an enzyme complex.We report now on the results of quantitative analyses of the carotenoids (lycopene, y-carotene, and (-carotene) produced by such heterokaryons. They coincide with the expected result if the synthesis is organized by enzyme complexes operating as assembly lines, and permit the description of certain features of such complexes.
MATERIALS AND METHODSP. blakesleeanus, strain C2, carrying the mutation car-i (previously designated Alb. 5) produces a white myceliumn and white fruiting bodies and contains only about 1% of the usual concentration of (3-carotene and no other carotenoids.Strain C9, carrying mutation carR21 (previously R1) is bright red, because of lycopene accumulation (8). Both were isolated from wild type strain NRRL1555, sexual type (-)r after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (7).A heterokaryon C2 * C9, containing nuclei of both strainsr was constructed artificially. The nuclei of the heterokaryon are randomly distributed into multinucleate spores, which can give rise to either type of homokaryon or to heterokaryons of different nuclear proportions. A heterokaryon may be propagated with a constant nuclear proportion by plating a. small piece of young mycelium onto a new medium (7).Mycelia are grown on glucose-asparagine-yeast solid medium (9) at room temperature (18-23°C) under normal daylight conditions. For chemical analysis, the mycelium grown 4 days in a Petri dish is scraped off with a spatula, carefully cleaned with tweezers of bits of agar and young sporangiophores, and stored in the dark at -20°C if the analysis is not done immediately. The mycelium is thawed at room temperature, dried with filter paper, cut to small piec...