Efficient Heterologous Transformation of Chlamydomonas reinhardtii npq2 Mutant with the Zeaxanthin Epoxidase Gene Isolated and Characterized from Chlorella zofingiensis

Abstract

In the violaxanthin cycle, the violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the inter-conversion between violaxanthin and zeaxanthin in both plants and green algae. The zeaxanthin epoxidase gene from the green microalga <em>Chlorella zofingiensis</em> (<em>Czzep</em>) has been isolated<em>. </em>This gene encodes a polypeptide of 596 amino acids. A single copy of <em>Czzep</em> has been found in the <em>C. zofingiensis</em> genome by Southern blot analysis. qPCR analysis has shown that transcript levels of <em>Czzep</em> were increased after zeaxanthin formation under high light conditions. The functionality of <em>Czzep</em> gene by heterologous genetic complementation in the <em>Chlamydomonas</em> mutant <em>npq2</em>, which lacks zeaxanthin epoxidase<strong> (</strong>ZEP) activity and accumulates zeaxanthin in all conditions, was analyzed. The <em>Czzep</em> gene was adequately inserted in the pSI105 vector and expressed in <em>npq2</em>. The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm). These results show that <em>Chlamydomonas</em> can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.