Identification and functional analysis of a phytoene desaturase gene from the extremely radioresistant bacterium Deinococcus radiodurans.
The phytoene-related desaturases are the key enzymes in the carotenoid biosynthetic pathway. The gene encoding phytoene desaturase in the deinoxanthin synthesis pathway of Deinococcus radiodurans was identified and characterized. Two putative phytoene desaturase homologues (DR0861 and DR0810) were identified by analysis of conserved amino acid regions, and the former displayed the highest identity (68 %) with phytoene desaturase of the cyanobacterium Gloeobacter violaceus. DR0861 gene knockout and dinucleotide-binding motif deletion resulted in the arrest of lycopene synthesis and the accumulation of phytoene. The colourless DR0861 knockout mutant became more sensitive to acute ionizing radiation and oxygen stress. Complementation of the mutant with a heterologous or homologous gene restored its pigment and resistance. The desaturase activity of DR0861 (crtI) was further confirmed by the assay of enzyme activity in vitro and heterologous expression in Escherichia coli containing crtE and crtB genes (responsible for phytoene synthesis) from Erwinia uredovora. In addition, the amount of lycopene synthesis in E. coli resulting from the expression of crtI from D. radiodurans was determined, and this had significant dose-dependent effects on the survival rate of E. coli exposed to hydrogen peroxide and ionizing radiation.
Xu
Zhenjian
Z
Institute of Nuclear-Agricultural Sciences, Zhejiang University, 310029 Hangzhou, China.
Tian
Bing
B
Institute of Nuclear-Agricultural Sciences, Zhejiang University, 310029 Hangzhou, China.
Sun
Zongtao
Z
Institute of Nuclear-Agricultural Sciences, Zhejiang University, 310029 Hangzhou, China.
Lin
Jun
J
Institute of Nuclear-Agricultural Sciences, Zhejiang University, 310029 Hangzhou, China.
Hua
Yuejin
Y
Institute of Nuclear-Agricultural Sciences, Zhejiang University, 310029 Hangzhou, China.
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002338:Carotenoids / Q000096:biosynthesis; D019295:Computational Biology; D017124:Conserved Sequence; D000458:Cyanobacteria / Q000235:genetics; D034301:Deinococcus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D004885:Erwinia / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D017353:Gene Deletion; D005816:Genetic Complementation Test; D000077276:Lycopene; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016877:Oxidants / Q000494:pharmacology; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D010860:Pigments, Biological / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D014466:Ultraviolet Rays
D000900:Anti-Bacterial Agents; D016877:Oxidants; D010860:Pigments, Biological; D002338:Carotenoids; D010088:Oxidoreductases; C022398:phytoene dehydrogenase; D000077276:Lycopene