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 photosynthetic membrane in all known oxygenic photosynthetic organisms. These yellow pigments serve the vital role of protecting against potentially 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 carotenoid lycopene to the bicyclic molecule beta-carotene. The gene was identified through the use of an experimental herbicide, 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA), that prevents the cyclization of lycopene in plants and cyanobacteria. Chemically-induced mutants of the cyanobacterium Synechococcus sp. PCC7942 were selected for resistance to MPTA, and a mutation responsible for this resistance was mapped to a genomic DNA region of 200 bp by genetic complementation of the resistance in wild-type cells. A 1.5 kb genomic DNA fragment containing this MPTA-resistance mutation was expressed in a lycopene-accumulating strain of Escherichia coli. The conversion of lycopene to beta-carotene in these cells demonstrated that this fragment encodes the enzyme lycopene cyclase. The results indicate that a single gene product, designated lcy, catalyzes both of the cyclization reactions that are required to produce beta-carotene from lycopene, and prove that this enzyme 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.
Cunningham
F X
FX
Department of Botany, University of Maryland, College Park 20742.
Chamovitz
D
D
Misawa
N
N
Gantt
E
E
Hirschberg
J
J
D002338:Carotenoids / Q000096:biosynthesis* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000235:genetics; D005021:Ethylamines / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D019753:Intramolecular Lyases; D007535:Isomerases / Q000235:genetics* / Q000378:metabolism; D000077276:Lycopene; D009154:Mutation; D010957:Plasmids; D014162:Transfection / Q000235:genetics; D019207:beta Carotene
D004269:DNA, Bacterial; D005021:Ethylamines; D019207:beta Carotene; C082524:2-(4-methylphenoxy)triethylamine; D002338:Carotenoids; D007535:Isomerases; D019753:Intramolecular Lyases; C070925:lycopene cyclase-isomerase; D000077276:Lycopene