[Cloning and analysis of the gene encoding lycopene epsilon cyclase in Chlorella protothecoides CS-41].
OBJECTIVE
Lycopene epsilon cyclase (LCYE) is the key enzyme in the lutein synthesis pathway and catalyses linear lycopene to form cyclic epsilon-carotene, a presucursor of lutein. We aimed to clone the full-length cDNA of LCYE gene from Chlorella protothecoides CS-41, to predict the functional sites and the three-dimensional structure of LCYE through bioinformatics analysis and to confirm its activities and functions.


METHODS
We used RACE (rapid-amplification of cDNA ends) essay and RT-PCR for the cloning of the full-length cDNA of LCYE from C. protothecoides CS-41. The online software such as PredictProtein, Pfam HMMs and Swiss-Model were used in bioinformatics analysis of the amino acid sequence of LCYE protein. We constructed the expression vector for LCYE gene with pET-28a(+) and transformed into Escherichia coli BL21 (DE3). Furthermore, the E. coli strain containing the pAC-LYC plasmid which could accumulate lycopene was used for the functional confirmation of LCYE from C. protothecoides CS-41.


RESULTS
A 2107 bp cDNA (GenBank Accession No. FJ752528) sequence was cloned with 1731 bp open reading frame, encoding a putative LCYE, from C. protothecoides CS-41. Homology studies showed that the deduced amino acid sequence of LCYE gene had a significant similarity with the corresponding sequences of other green algae and higher plants. It shared the highest sequence identity, up to 67%, with the LCYE gene from Chlamydomonas reinhardtii. One typical lycopene cyclase protein domain (Pfam05834) was predicted between the 48th-459th amino acid. In addition, the sequence between 261th -284th was one typical conserved lycopene cyclase protein motif. The SDS-PAGE result showed that the LCYE gene was overexpressed in Escherichia coli BL21 (DE3) after the addition of IPTG. The prokaryotically expressed LCYE protein was able to transfer the color of the E. coli strain containing the pAC-LYC plasmid from pink to yellow.


CONCLUSIONS
The full-length cDNA sequence of LCYE gene was successfully cloned with the size of 2107 bp. Several typical motifs were found and the three-dimensional structure of LCYE was constructed from Bioinformatics analysis. The generated phylogenetic tree showed the closest relationship between C. protothecoides CS-41 and C. reinhardtii among the listed organisms. Finally, the expression product of the LCYE gene cloned in the study was confirmed to hold the function and activity of lycopene epsilon cyclase.
Li
Ting
T
Department of Food Science & Technology and Bor Luh Food Safety Center, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200240, China. liting8848@hotmail.com
Shi
Chunlei
C
Gan
Zhibing
Z
Shi
Xianming
X
D020418:Algal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002708:Chlorella / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D019753:Intramolecular Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid
D020418:Algal Proteins; D019753:Intramolecular Lyases; C070925:lycopene cyclase-isomerase