[Cloning of Blakeslea trispora carRA gene by PCR-driven overlap extension and construction of an activity detection system].
Blakeslea trispora CarRA has both lycopene cyclase and phytoene synthase activity. In order to analyze the double functional activity of CarRA proteins and to detect the active sites of lycopene cyclase, we constructed two detection systems in Escherichia coli by color complementary. Through PCR-driven overlap extension we got carRA gene cDNA, then constructed prokaryotes expression vector pET28a-carRA. pET28a-carRA with plasmid pAC-LYC carrying crtl/crtB/crtE gene clusters were co-transformed to BL21(DE3) to validate lycopene cyclase activity. We constructed the plasmid pAC-LYC delta (crtB) carrying crtl/crtE gene clusters, then co-transtormed them with pET28a-carRA to BL21(DE3) to validate phytoene synthase activity. Based on color complementary, and HPLC analysis of metabolites, we confirmed that the CarRA protein activity detection system was reliable. Our study provides a screening model for specific mutation of lycopene cyclase without affecting phytoene synthase activity.
Key Laboratory of Microbial Diversity Research and Application of Hebei Province, College of Life Sciences, Hebei University, Baoding 071002, China.
Hui
H
Tang
Nan
N
Shi
Miao
M
Yu
Long
L
Liu
Jing
J
Liu
Ying
Y
Jia
Hongyan
H
Niu
Liping
L
Zhang
D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism*; D002338:Carotenoids / Q000096:biosynthesis*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase; D019753:Intramolecular Lyases / Q000235:genetics / Q000378:metabolism*; D000077276:Lycopene; D009090:Mucorales / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D016133:Polymerase Chain Reaction
D018076:DNA, Complementary; D005656:Fungal Proteins; D002338:Carotenoids; D019883:Alkyl and Aryl Transferases; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase; D019753:Intramolecular Lyases; C070925:lycopene cyclase-isomerase; D000077276:Lycopene