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 Chlorella zofingiensis (Czzep) has been isolated. This gene encodes a polypeptide of 596 amino acids. A single copy of Czzep has been found in the C. zofingiensis genome by Southern blot analysis. qPCR analysis has shown that transcript levels of Czzep were increased after zeaxanthin formation under high light conditions. The functionality of Czzep gene by heterologous genetic complementation in the Chlamydomonas mutant npq2, which lacks zeaxanthin epoxidase (ZEP) activity and accumulates zeaxanthin in all conditions, was analyzed. The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2. 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 Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.
Keywords: Chlamydomonas reinhardtii; Chlorella zofingiensis; carotenoids; violaxanthin cycle; zeaxanthin epoxidase.