Biosynthesis of @Carotene by Cell-free Extracts of Phycomyces blakesleeanus

Most information on the biosynthesis of carotenoids is from experiments with fungi, especially Phycomyces blakesleeanus and Mucor hiemalis; with bacteria, notably the photosynthetic bacteria, Rhodospirillum rubrum and Rhodopseudomonas spheroides; and with carrot roots and homogenates, tomato fruit and homogenates, and algae and etiolated seedlings. Synthesis in vitro of carotenoids by the above has been demonstrated in carrot root homogenates, tomato fruit homogenates, and, in one instance, extracts of carrot roots (1, 2). The formation of carotenoids from small molecules uch as acetate and mevalonic acid has been repeatedly demonstrated in growing microorganisms and in plant tissues (3-5). Since whole cells are generally impermeable to the transport of large molecules, which may be important intermediates in the synthesis, the development of an effective cell-free system capable of forming carotenoids appears to be essential for a more detailed study of the biosynthetic mechanisms involved. Goodwin reported in a number of reviews that cell-free extracts of carrot roots are capable of synthesizing p-carotene, but he has not published details of this system (1). It would be of interest to be able to compare the cofactor requirements in the Phycomyces system with those of Goodwin. Shneour and Zabin, in reporting the synthesis of lycopene in homogenates of tomatoes, found that their system would incorporate activity from mevalonic acid but not from acetate (6). This system was not active if the cell debris were removed by centrifugation, although the authors state that no intact cells were visible when examined microscopically. Cofactor requirements for synthesis of lycopene from mevalonic acid were adenosine triphosphate, diphosphopyridine nucleotide, triphosphopyridine nucleotide, glutathione and Mn++. In thii paper we report on the partially purified cell-free enzyme system obtained from Phycomyces blakesleeanus, which is capable of synthesizing p-carotene from sodium acetate, hydroxymethylglutarate, and mevalonic acid. The isoprene labeling pattern in the p-carotene obtained from the system is similar to that observed in growing microorganisms and in plint tissue.