Engineering of the carotenoid pathway in Xanthophyllomyces dendrorhous leading to the synthesis of zeaxanthin.

Zeaxanthin is an essential nutrient for prevention of macular degeneration. However, it is limited in our diet. For the production of zeaxanthin, we have engineered zeaxanthin synthesis into a carotenoid mutant of Xanthophyllomyces dendrorhous which is blocked in astaxanthin synthesis and accumulates β-carotene instead. Two strategies were followed to reach high-yield zeaxanthin synthesis. Total carotenoid synthesis was increased by over-expression of genes HMGR, crtE, and crtYB encoding for limiting enzymes in the pathway leading to and into carotenoid biosynthesis. Then bacterial genes crtZ were used to extend the pathway from β-carotene to zeaxanthin in this mutant. The increase of total carotenoids and the formation of zeaxanthin is dependent on the number of gene copies of crtYB and crtZ integrated into the X. dendrorhous upon transformation. The highest zeaxanthin content around 500 μg/g dw was reached by shaking flask cultures after codon optimization of crtZ for Xanthophyllomyces. Stabilization of carotenoid and zeaxanthin formation in the final transformant in the absence of selection agents was achieved after passing through a sexual cycle and germination of basidiospores. The values for the transformant before and after stabilization were very similar resembling about 70 % of total carotenoids and corresponding to a conversion rate of 80 % for hydroxylation of β-carotene to zeaxanthin. The stabilized transformant allowed experimental small-scale fermentation yielding X. dendrorhous cells with a zeaxanthin content similar to the shaking flask cultures. Our result demonstrates the potential of X. dendrorhous for its development as a zeaxanthin producer and its suitability for large-scale fermentation.