Xanthophyllomyces dendrorhous, a Versatile Platform for the Production of Carotenoids and Other Acetyl-CoA-Derived Compounds.

Xanthophyllomyces dendrorhous (with Phaffia rhodozyma as its anamorphic state) is a basidiomycetous, moderately psychrophilic, red yeast belonging to the Cystofilobasidiales. Its red pigmentation is caused by the accumulation of astaxanthin, which is a unique feature among fungi. The present chapter reviews astaxanthin biosynthesis and acetyl-CoA metabolism in X. dendrorhous and describes the construction of a versatile platform for the production of carotenoids, such as astaxanthin, and other acetyl-CoA-derived compounds including fatty acids by using this fungus.

Genetic modification of the carotenoid pathway in the red yeast Xanthophyllomyces dendrorhous: Engineering of a high-yield zeaxanthin strain.

The red yeast Xanthophyllomyces dendrorhous was genetically engineered for high-yield accumulation of the carotenoid zeaxanthin. Initially, an astaxanthin hyper-producing mutant was used to generate a ß-carotene synthesizing transformant by inactivation of the astaxanthin synthase gene. Subsequently, a bacterial ß-carotene hydroxylase gene was genome integrated to establish ß-carotene to zeaxanthin conversion. Crucial for efficient zeaxanthin formation was the rate of this hydroxylation which was related to the number of integrated gene copies. Two strategies were followed to get multiple integrations, either random integration into the ribosomal DNA which resulted in a maximum copy number of 10, or directly integration of a total of 8 copies into both alleles of the astaxanthin synthase gene. Combining both procedures with additional insertion of the gene to enhance expression of the carotenogenesis limiting phytoene synthase, a transformant reaching a high level of zeaxanthin of 5.2 mg/g dw was finally generated. The application of pentose sugars including xylose as substrates for X. dendrorhous which avoids the inhibitory Crab-tree effect of glucose is favorable for carotenogenesis allowing the replacement of glucose by a hydrolysate of the waste product hemicellulose which is rich in xylose demonstrating ithe effectiveness as a sustainable and cost-efficient alternative for high-yield zeaxanthin formation.

Development of Xanthophyllomyces dendrorhous as a production system for the colorless carotene phytoene.

Phytoene is a colorless carotenoid with increasing economic potential for skin care but with limited availability. The red yeast Xanthophyllomyces dendrorhous which has previously been used as a production platform for carotenoids was engineered as a prototype for the yield of this carotene. Phytoene was accumulated by prevention of its metabolization by desaturation in the carotenoid pathway. In a first step, the phytoene desaturase gene crtI was disrupted by insertion of a hygromycin-resistance gene. Most of the resulting transformants were heterozygote for intact and inactivated crtI. Upon re-cultivation of this orange transformants under selection pressure, white colonies homozygote for disrupted crtI were obtained. In contrast to reddish wild-type, the orange transformants contained colored carotenoids together with phytoene whereas the homozygote transformant synthesized phytoene exclusively. This targeted mutagenesis approach was first tested with the wild type and then applied to a high-yield carotenoid synthesizing X. dendrorhous mutant. In a second step, precursor supply for phytoene synthesis was enhanced by over-expression of the genes HMGR, crtE and crtYB which encode limiting enzymes of the pathway. The combination of this engineering approaches resulted in a phytoene producing X. dendrorhous strain which accumulated 7.5mg/g dw in shaking cultures. Finally, experimental small scale fermenter studies demonstrated continuous growth of this strain during fermentation and stable phytoene production without selection pressure. This fermenter culture contained the highest phytoene content ever reached by any organism with more than 10mg/g dw.

Combinatorial Biosynthesis of Novel Multi-Hydroxy Carotenoids in the Red Yeast Xanthophyllomyces dendrorhous.

The red yeast Xanthophyllomyces dendrorhous is an established platform for the synthesis of carotenoids. It was used for the generation of novel multi oxygenated carotenoid structures. This was achieved by a combinatorial approach starting with the selection of a ß-carotene accumulating mutant, stepwise pathway engineering by integration of three microbial genes into the genome and finally the chemical reduction of the resulting 4,4'-diketo-nostoxanthin (2,3,2',3'-tetrahydroxy-4,4'-diketo-ß-carotene) and 4-keto-nostoxanthin (2,3,2',3'-tetrahydroxy-4-monoketo-ß-carotene). Both keto carotenoids and the resulting 4,4'-dihydroxy-nostoxanthin (2,3,4,2',3',4'-hexahydroxy-ß-carotene) and 4-hydroxy-nostoxanthin (2,3,4,2'3'-pentahydroxy-ß-carotene) were separated by high-performance liquid chromatography (HPLC) and analyzed by mass spectrometry. Their molecular masses and fragmentation patterns allowed the unequivocal identification of all four carotenoids.

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 ß-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 ß-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 ß-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.

Multiple transformation with the crtYB gene of the limiting enzyme increased carotenoid synthesis and generated novel derivatives in Xanthophyllomyces dendrorhous.

Xanthophyllomces dendrorhous (in asexual state named as Phaffia rhodozyma) is a fungus which produces astaxanthin, a high value carotenoid used in aquafarming. Genetic pathway engineering is one of several steps to increase the astaxanthin yield. The limiting enzyme of the carotenoid pathway is phytoene synthase. Integration plasmids were constructed for transformation with up to three copies of the crtYB gene. Upon stepwise transformation, the copy numbers of crtYB was continuously increased leading to an almost saturated level of phytoene synthase as indicated by total carotenoid content. Several carotenoid intermediates accumulated which were absent in the wild type. Some of them are substrates and intermediates of astaxanthin synthase. They could be further converted into astaxanthin by additional transformation with the astaxanthin synthase gene. However, three intermediates exhibited an unusual optical absorbance spectrum not found before. These novel keto carotenoid were identified by HPLC co-chromatography with reference compounds generated in Escherichia coli and one of them 3-HO-4-keto-7',8'-dihydro-ß-carotene additionally by NMR spectroscopy. The others were 4-keto-ß-zeacarotene and 4-keto-7',8'-dihydro-ß-carotene. A biosynthesis pathway with their origin from neurosporene and the reason for their synthesis especially in our transformants has been discussed.