Metabolic Engineering of the Carotenoid Biosynthetic Pathway in the Yeast Xanthophyllomyces dendrorhous ( Phaffia rhodozyma )

Abstract

The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a ␤-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the ␤-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono-and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via ␤-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3-4-didehydro-␤--caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.

Astaxanthin and Related Xanthophylls

“…Although the overexpression of the crtYB gene (which encodes the phytoene-lycopene synthase) led to an increase in the overall carotenoid synthesis, it was mainly due to higher amounts of β(beta)-carotene and echinenone with an unaffected (or even lower) astaxanthin content observed [ 127 ]. In contrast, the overexpression of the crtI gene (phytoene desaturase encoding gene) decreased the total carotenoid production and varied its composition.…”

Production of Enriched Biomass by Carotenogenic Yeasts - Application of Whole-Cell Yeast Biomass to Production of Pigments and Other Lipid Compounds

“…In fact, addition of mevalonate to a culture of X. dendrourhous stimulated both astaxanthin and total carotenoid biosynthesis four times (from 0.18 to 0.76 mg/g and from 0.27 to 1.1 mg/g dry cells, respectively). This indicates that the conversion of HMG-CoA to mevalonate by HMG-CoA reductase is a potential bottleneck on the road to modified strains with higher astaxanthin content (Verdoes et al, 2003).…”

“…Fusarium sporotrichioides was genetically modified for lycopen production by redirecting of the isoprenoid pathway toward the synthesis of carotenoids and introducing genes from the bacterium Erwinia uredovora (Leathers et al, 2004). Carotenoid biosynthetic pathway of astaxanthin producers of Phaffia/Xanthophyllomyces strains has also been engineered and several genes, such as phytoene desaturase, isopentenyl diphosphate isomerase and epoxide hydrolase were isolated and expressed in E. coli (Verdoes et al, 2003;Lukacz, 2006).…”

High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous

“…xanthin biosynthesis pathway (1,7,35). The genes involved in ␤-carotene production in X. dendrorhous have been cloned previously (33,34).…”

“…Subsequently, two cyclization reactions catalyzed by CrtYB result in the conversion of lycopene into ␥-carotene and finally into ␤-carotene. X. dendrorhous mutants with higher carotenoid production capacities have been obtained by chemical mutagenesis (1,7) or by recombinant DNA technology (35,39).…”