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Mar Biotechnol (2009) 11:1723 DOI 10.1007/s10126-008-9116-7
ORIGINAL ARTICLE
Identification of a Novel C20-elongase Gene from the Marine Microalgae Pavlova viridis and its Expression in Escherichia coli
Yan Niu & Jian Kong & Longyun Fu & Jing Yang & Yi Xu
Received: 5 December 2007 /Accepted: 30 April 2008 /Published online: 24 July 2008 # Springer Science + Business Media, LLC 2008
Abstract Pavlova viridis, a species of a unicellular marine microalgae, is rich in the very-long-chain polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). A new elongase gene (elkj), with high identity with a functionally characterized C20-elongase of Pavlova lutheri, was isolated via reverse transcriptase-polymerase chain reaction using the primers designed from conserved motifs and 5/3 rapid amplification of cDNA ends. The coding region of 314 amino acids predicted a protein of 34 kDa, which contained seven transmembrane domains with its C-terminal in the cytoplasm and located in the endoplasmic reticulum. The expression of ELKJ in Escherichia coli was carried out by using green fluorescent protein as an indicator, suggesting the correct insertion in cytoplasmic membrane. Functional analysis demonstrated that elkj encoded a C20-elongase that mediated the elongation of EPA into docosapentaenoic acid (22:5n-3), confirming the two-step conversion from EPA to DHA in marine microalga.
Keywords Pavlova viridis . Elongase . Membrane protein . Green fluorescent protein
Introduction
The long-chain 53-polyunsaturated fatty acids (53-PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are essential components of membrane lipids. DHA is found in high proportion in neuronal membranes and the external segments of photo-receptors in retina (Neuringer 2000). The deficiency of
DHA has been proved to be associated with cognitive decline and the onset of Alzheimers disease in adults (Tully et al. 2003). In infants, DHA has been indicated to be vital for proper visual and neurological development. DHA cannot be produced in sufficient amount to meet metabolic demands of the human body and, therefore, must be obtained from dietary sources. The major dietary sources of DHA are oils from marine fish, which obtain most of the 53-PUFAs by consumption of marine microalgae. The lack of fish sources and the increase in demand have raised interest in obtaining DHA by genetic manipulation.
In mammals, the conversion of EPA into...