Identification of a Novel C20-elongase Gene from the Marine Microalgae Pavlova viridis and its Expression in Escherichia coli

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.

Metabolic Engineering and Oil Supplementation of Physcomitrella patens for Activation of C22 Polyunsaturated Fatty Acid Production

“…The D 5 -elongation conversion efficiency in P. patens for x-6 PUFAs was higher than for x-3 PUFAs, probably due to lesser amount of EPA substrate in P. patens. Recently, the cotton D 12 -desaturase (FAD2-4)-and marine microalgae Pavlova viridis C20-elongase-GFP fusion polypeptides appeared to be functionally expressed in transgenic Arabidopsis plants and E. coli, respectively, and were shown to localized in the cytoplasmic membrane [28,29]. Our results also showed that when PsELO5 was attached to the C-terminal of GFP6, PsELO5 function was stronger than with a non-fusion protein.…”

Key Enzymes in Fatty Acid Synthesis Pathway for Bioactive Lipids Biosynthesis

“…5-elongase is a kind of fatty acid elongase with C20 fatty acid as substrate which catalyze arachidonic acid (AA, C20:4 5,8,11,14 ) and eicosapentaenoic acid (EPA, C20:4 5,8,11,14,17 ) to Docosatetraenoic acid (DTA, C22:4 7,10,13,16 ) and Docosapentaenoic acid (DPA, C22:5 7,10,13,16,19 ), respectively. Employing fluorescent protein as an indicator, Niu et al (87) found the 5-elongase from Pavlova viridis located in the endoplasmic reticulum. Robert and his colleague identified a -5 elongase from Pavlova salina and characterized the exclusive elongase function for EPA.…”

Heterologous overexpression of a novel delta-4 desaturase gene from the marine microalga Pavlova viridis in Escherichia coli as a Mistic fusion

“…As a matter of fact, the OD 600 of the culture of the recombinant strain did not appear to increase any more under the induction condition compared to the control group. In the case of the use of the green fluorescent protein (GFP) fused to the carbon terminus of pkjDes4 as a reporter, which was used to detect the expression of C-20 elongase of the same species (Niu et al 2009), still no signal of green fluorescence was detected (data not shown). Considering the high sensitivity of GFP detection, it could be concluded that the toxicity of the host cells brought by the targeting, translocation or membrane insertion of pkjDes4 was too severe to yield any detectable recombinant proteins.…”

“…Its C20-elongase gene has been cloned with 5 0 and 3 0 RACE and functionally characterized in our lab (Niu et al 2009). In this study, we isolated the other key gene participating in EPA-DHA conversion, pkjDes4, largely considered as the rate-limiting step in the reaction.…”