CLONING AND FUNCTIONAL EXPRESSION IN ESCHERICHIA-COLI OF A CYANOBACTERIAL GENE FOR LYCOPENE CYCLASE, THE ENZYME THAT CATALYZES THE BIOSYNTHESIS OF BETA-CAROTENE

Carotenoids with cyclic end groups are essential components of the pho tosynthetic membrane in all known oxygenic photosynthetic organisms. T hese yellow pigments serve the vital role of protecting against potent ially lethal photo-oxidative damage. Many of the enzymes and genes of the carotenoid biosynthetic pathway in cyanobacteria, algae and plants remain to be isolated or identified. We have cloned a cyanobacterial gene encoding lycopene cyclase, an enzyme that converts the acyclic ca rotenoid lycopene to the bicyclic molecule beta-carotene. The gene was identified through the use of an experimental herbicide, 244-methylph enoxy)triethylamine hydrochloride (MPTA), that prevents the cyclizatio n of lycopene in plants and cyanobacteria. Chemically-induced mutants of the cyanobacterium Synechococcus sp. PCC7942 were selected for resi stance to MPTA, and a mutation responsible for this resistance was map ped to a genomic DNA region of 200 bp by genetic complementation of th e resistance in wild-type cells. A 1.5 kb genomic DNA fragment contain ing this MPTA-resistance mutation was expressed in a lycopene-accumula ting strain of Escherichia coli. The conversion of lycopene to beta-ca rotene in these cells demonstrated that this fragment encodes the enzy me lycopene cyclase. The results indicate that a single gene product, designated lcy, catalyzes both of the cyclization reactions that are r equired to produce beta-carotene from lycopene, and prove that this en zyme is a target site of the herbicide MPTA. The cloned cyanobacterial lcy gene hybridized well with genomic DNA from eukaryotic algae, thus it will enable the identification and cloning of homologous genes for lycopene cyclase in algae and plants.