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.