Cloning And Characterization Of The Lcye Gene Encoding Lycopene Epsilon Cyclase In Chlorella Protothecoides

Lutein is one of xanthophylls which are a group of compounds that are oxygenated derivatives of carotenoids. Natural lutein demonstrates high performance in anti-oxidation effect. Chlorella is noted for its high cellular lutein content, which makes it a potential resource for lutein production. Comparing to other lutein resources, Chlorella has many advantages such as the high specific growth rate and the ability of growing heterotrophically. Most researches focused on the optimization of cultivation conditions to improve lutein productivity; however, few researches used metabolic engineering technology to achieve this aim. The objective of this study is to clone the full-length cDNA of LCYE gene from Chlorella protothecoides CS-41 and to preliminarily understand the regulatory expression mechanism of LCYE at the molecular level by bioinformatics analysis. The following aspects were included in this study.1,A full-length cDNA (GenBank Accession No.FJ752528)of LCYE was cloned from Chlorella protothecoides CS-41 by RACE (rapid-amplification of cDNA ends) and RT-PCR, which is a 2107-bp sequence. It was found that there was a 1731-bp open reading frame by ORF finder, and that this gene encodes a putative LCYE from Chlorella protothecoides CS-41.2,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 similarity, up to 67%, with the LCYE of Chlamydomonas reinhardtii. It was predicted that one typical lycopene cyclase protein domain (Pfam05834) was located between the 48th -459th amino acid. Several typical motifs for all cyclases were found by the alignment analysis of the LCYE amino acid sequences: one Dinucleotide-binding site at the N-terminus, one Cyclase motif 1, and one Predicted TM helix at the C-terminus. The anaysis of phylogenetic tree showed the closest relationship between Chlorella protothecoides CS-41 and Chlamydomonas reinhardtii.3,Taqman real-time PCR and HPLC (high performance liquid chromatography) were respectively used to determine the expression level of LCYE gene and the lutein content of different cultures, and it was indicated that the expression of LCYE gene correlated with lutein content in different cultivation conditions.