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

Abstract

SummaryMixed cultures of strains of opposite sex of the Mucorales produce trisporic acids and other compounds arising from cleavage of b-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 b-carotene-producing Escherichia coli cells led to the formation of b-apo-12Ј-carotenal, a C 25 cleavage product of b-carotene. Joint expression of both genes in the same in vivo system resulted in the production of b-apo-13-carotenone, a C18 fragment. In vitro, AcaA cleaved b-apo-12Ј-carotenal into b-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 b-carotene at its C11Ј-C12Ј double bond by CarS and the cleavage of the resulting C25-fragment at its C13-14 double bond by AcaA.

The Oxygenase CAO-1 of Neurospora crassa Is a Resveratrol Cleavage Enzyme

“…2). The enzymes CAO-2 of Neurospora and CarT of F. fujikuroi are clustered in the phylogenetic tree with CarS and AcaA, two CCOs recently discovered in the zygomycete P. blakesleeanus (20,21). CAO-2 and CarT cleave the monocyclic carotene torulene, and CarS cleaves ␤-carotene to produce the substrate for AcaA, ␤-apo-12=carotenal.…”

“…As a further approach to test a possible activity of CAO-1 on carotenoids, crude lysates of thioredoxin-CAO-1-overexpressing cells (described below) were incubated in vitro with different carotenoid substrates that are cleaved by other CCO enzymes (20,31). No traces of enzymatic activity could be detected after HPLC separation of the carotenoids extracted from the reaction mixtures (see Fig.…”

“…For instance, the zygomycete Phycomyces blakesleeanus employs two CCOs, CarS and AcaA, to sequentially convert ␤-carotene into the C 18 ketone ␤-apo-13-carotenone via the C 25 intermediate ␤-apo-12=-carotenal (20,21) in the pathway leading to the trisporic acid pheromones. The ascomycetes Neurospora crassa (referred to hereinafter as Neurospora) and Fusarium fujikuroi produce neurosporaxanthin, a C 35 apocarotenoic acid.…”

On the substrate‐ and stereospecificity of the plant carotenoid cleavage dioxygenase 7

“…SLs belong to the apocarotenoids [15][16][17][18], a group of carotenoidderived, physiologically important compounds that includes retinoids, the phytohormone abscisic acid and the fungal pheromone trisporic acid [19]. The precursors of SLs, the carotenoids, are isoprenoid pigments with an extended conjugated double bond system synthesized by all photosynthetic organisms and many heterotrophic bacteria and fungi [20][21][22][23].…”

Early and Late Trisporoids Differentially Regulate β-Carotene Production and Gene Transcript Levels in the Mucoralean Fungi Blakeslea trispora and Mucor mucedo

“…Little is known about the physiological functions of enzymes or the products of ␤-carotene cleavage and trisporic acid biogenesis within the Mucorales. The hypothetical biosynthetic pathway in P. blakesleeanus commences with oxidative cleavage of ␤-carotene at the C 11= OC 12= as the caroteneoid cleavage dioxygenase (CarS) releases ␤-apo-12=-carotenal (C 25 ), which further cleaves to the first C 18 trisporoid (␤-apo-13-carotenone) D'orenone at the C 13 OC 14 bond by apocarotenoid cleavage oxygenase (AcaA) (14). CarS mutants in Phycomyces fail to produce those apocarotenoids present in the wild type (15).…”