Cleavage oxygenases for the biosynthesis of trisporoids and other apocarotenoids in Phycomyces.

Mixed cultures of strains of opposite sex of the Mucorales produce trisporic acids and other compounds arising from cleavage of β-carotene, some of which act as signals in the mating process. The genome of Phycomyces blakesleeanus contains five sequences akin to those of verified carotenoid cleavage oxygenases. All five are transcribed, three of them have the sequence traits that are considered essential for activity, and we have discovered the reactions catalysed by the products of two of them, genes carS and acaA. The transcripts of carS became more abundant in the course of mating, and its expression in β-carotene-producing Escherichia coli cells led to the formation of β-apo-12'-carotenal, a C₂₅ cleavage product of β-carotene. Joint expression of both genes in the same in vivo system resulted in the production of β-apo-13-carotenone, a C₁₈ fragment. In vitro, AcaA cleaved β-apo-12'-carotenal into β-apo-13-carotenone and was active on other apocarotenoid substrates. According to these and other results, the first reactions in the apocarotenoid pathway of Phycomyces are the cleavage of β-carotene at its C11'-C12' double bond by CarS and the cleavage of the resulting C₂₅-fragment at its C13-14 double bond by AcaA.