Functional complementation in Escherichia coli of different phytoene desaturase genes and analysis of accumulated carotenes.
Three different phytoene desaturase genes, from Rhodobacter capsulatus, Erwinia uredovora, and Synechococcus PCC 7942, have been functionally complemented with a gene construct from E. uredovora which encodes all enzymes responsible for formation of 15-cis phytoene in Escherichia coli. As indicated by the contrasting reaction products detected in the pigmented E. coli cells after co-transformation, a wide functional diversity of these three different types of phytoene desaturases can be concluded. The carotenes formed by the phytoene desaturase from R. capsulatus were trans-neurosporene with three additional double bonds and two cis isomers. Furthermore, small amounts of three zeta-carotene isomers (2 double bonds more than phytoene) and phytofluene (15-cis and all-trans with + 1 double bond) were detected as intermediates. When the subsequent genes from E. uredovora which encode for lycopene cyclase and beta-carotene hydroxylase were present, neurosporene, the phytoene desaturase product of R. capsulatus, was subsequently converted to the monocyclic beta-zeacarotene and its monohydroxylation product. The most abundant carotene resulting from phytoene desaturation by the E. uredovora enzyme was trans-lycopene together with a cis isomer. In addition, bisdehydrolycopene was also formed. The reaction products of Synechococcus phytoene desaturase were two cis isomers of zeta-carotene and only small amounts of trans-zeta-carotene including 15-cis. The I50 values for flurtamone and diphenylamine to inhibit phytoene desaturation were determined and differential inhibition was observed for diphenylamine.
Linden
H
H
Lehrstuhl für Physiologie und Biochemie der Pflanzen, Universität Konstanz, Bundesrepublik Deutschland.
Misawa
N
N
Chamovitz
D
D
Pecker
I
I
Hirschberg
J
J
Sandmann
G
G
D002338:Carotenoids / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics*; D004885:Erwinia / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D007700:Kinetics; D010088:Oxidoreductases / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000201:enzymology / Q000235:genetics*
D011994:Recombinant Proteins; D002338:Carotenoids; D010088:Oxidoreductases; C022398:phytoene dehydrogenase