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

Catalina Sanz,María Isabel Álvarez,Ernesto P. Benito,Antonio Velayos,Arturo P. Eslava,Michael Freitag

doi:10.1371/journal.pone.0023102

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.

Highlights

Carotenoids are natural pigments widely distributed that contribute to the color of many plants and animals and play a major role in photoprotection
The biosynthetic pathway of all C40 carotenoids starts with the condensation of two geranylgeranyl pyrophosphate (GGPP) molecules to form the colorless compound phytoene, a step catalyzed by the enzyme phytoene synthase
In order to confirm that the carRA gene codes for an enzyme with lycopene cyclase and phytoene synthase activities, as the M. circinelloides carRP gene does, functional complementation in M. circinelloides was attempted

Summary

Introduction

Carotenoids are natural pigments widely distributed that contribute to the color of many plants and animals and play a major role in photoprotection. The biosynthetic pathway of all C40 carotenoids starts with the condensation of two geranylgeranyl pyrophosphate (GGPP) molecules to form the colorless compound phytoene, a step catalyzed by the enzyme phytoene synthase. From this point, the pathway can take different directions depending on the organism [5]. Two cyclizations at both ends of the lycopene molecule catalyzed by the enzyme lycopene cyclase give rise to ß-carotene, the end-product of the pathway (Figures 1 and 2) These enzymes are thought to be organized in an aggregate, formed by four and two units of phytoene dehydrogenase and lycopene cyclase, respectively, that works as an assembly line [6,7,8,9,10]

Methods

Results

Conclusion