Functional Analysis of the Phycomyces carRA Gene Encoding the Enzymes Phytoene Synthase and Lycopene Cyclase

Abstract

Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism.

The genome of the basal agaricomycete Xanthophyllomyces dendrorhous provides insights into the organization of its acetyl-CoA derived pathways and the evolution of Agaricomycotina

“…All carotenogenic fungi possess the crtYB and crtI genes. For example in Phycomyces blakesleeanus and Mucor circinelloides , both genes are found next to each other with a spacing of 1.4 or 0.5 kbp, respectively, but convergently transcribed [ 48 ] and in Fusarium fujikuroi , they are 0.6 kbp apart and transcribed together in the same direction [ 49 ].…”

Comparative Analysis of β-Carotene Production by Mucor circinelloides Strains CBS 277.49 and WJ11 under Light and Dark Conditions

“…This conversion is considered as the first bottleneck in mevalonate pathway, since carG was found to be co-regulated with the structural genes (carB and carRP) of the carotenoid biosynthesis pathway [18]. Genetic analysis of carotene mutants in P. blakesleeanus found only two structural genes, carB and carR, are responsible for the conversion of phytoene to β-carotene [19,20]. In M. circinelloides, these two genes are "carB (Phytoene dehydrogenase)" [21] and "carRP (phytoene synthase/lycopene cyclase)" [22], which carry out the conversion of Phytoene to lycopene and then from lycopene to β-carotene, respectively (Figure 1).…”

Carotenoids

“…In contrast to plants and bacteria, fungal phytoene synthase and cyclase enzymatic activities reside in two protein domains of a bifunctional enzyme encoded by a single gene. This dual activity was fi rstly discovered in the basidiomycete X. dendrorhous [ 33 ] and confi rmed in P. blakesleeanus by mutation analysis [ 34 ] and in M. circinelloides by partial deletion analysis [ 35 ], by complementation of different carRP mutants and by heterologous expression in engineered E. coli strains [ 36 ]. Therefore, only two genes encoding a phytoene synthase/cyclase and a phytoene dehydrogenase are needed to produce β-carotene from GGPP.…”