The red heterobasidiomycetous yeast Xanthophyllomyces dendrorhous (perfect state of Phaffia rhodozyma) contains a novel type of carotenoid biosynthetic enzyme. Its structural gene, designated crtYB, was isolated by functional complementation in a genetically modified, carotenogenic Escherichia coli strain. Expression studies in different carotenogenic E. coli strains demonstrated that the crt YB gene encodes a bifunctional protein involved both in synthesis of phytoene from geranylgeranyl diphosphate and in cyclisation of lycopene to beta-carotene. By sequence comparison with other phytoene synthases and complementation studies in E. coli with various deletion derivatives of the crtYB gene, the regions responsible for phytoene synthesis and lycopene cyclisation were localised within the protein.

No abstract

This review describes the different approaches that have been used to manipulate and improve carotenoid production in Xanthophyllomyces dendrorhous. The red yeast X. dendrorhous (formerly known as Phaffia rhodozyma) is one of the microbiological production systems for natural astaxanthin. Astaxanthin is applied in food and feed industry and can be used as a nutraceutical because of its strong antioxidant properties. However, the production levels of astaxanthin in wild-type isolates are rather low. To increase the astaxanthin content in X. dendrorhous, cultivation protocols have been optimized and astaxanthin-hyperproducing mutants have been obtained by screening of classically mutagenized X. dendrorhous strains. The knowledge about the regulation of carotenogenesis in X. dendrorhous is still limited in comparison to that in other carotenogenic fungi. The X. dendrorhous carotenogenic genes have been cloned and a X. dendrorhous transformation system has been developed. These tools allowed the directed genetic modification of the astaxanthin pathway in X. dendrorhous. The crtYB gene, encoding the bifunctional enzyme phytoene synthase/lycopene cyclase, was inactivated by insertion of a vector by single and double cross-over events, indicating that it is possible to generate specific carotenoid-biosynthetic mutants. Additionally, overexpression of crtYB resulted in the accumulation of beta-carotene and echinone, which indicates that the oxygenation reactions are rate-limiting in these recombinant strains. Furthermore, overexpression of the phytoene desaturase-encoding gene (crtI) showed an increase in monocyclic carotenoids such as torulene and HDCO (3-hydroxy-3',4'-didehydro-beta,-psi-carotene-4-one) and a decrease in bicyclic carotenoids such as echinone, beta-carotene and astaxanthin.

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.

Arabinoxylan arabinofuranohydrolase-D3 (AXHd3) from Bifidobacterium adolescentis releases only C3-linked arabinose residues from double-substituted xylose residues. A genomic library of B. adolescentis DSM20083 was screened for the presence of the axhD3 gene. Two plasmids were identified containing part of the axhD3 gene. The nucleotide sequences were combined and three open reading frames (ORFs) were found. The first ORF showed high homology with xylanases belonging to family 8 of the glycoside hydrolases and this gene was designated xylA. The second ORF was the axhD3 gene belonging to glycoside hydrolase family 43. The third (partial) ORF coded for a putative carboxylesterase. The axhD3 gene was cloned and expressed in Escherichia coli. Several substrates were employed in the biochemical characterization of recombinant AXHd3. The enzyme showed the highest activity toward wheat arabinoxylan oligosaccharides. In addition, beta-xylanase from Trichoderma sp. was able to degrade soluble wheat arabinoxylan polymer to a higher extent, after pretreatment with recombinant AXHd3. Arabinoxylan oligosaccharides incubated with a combination of recombinant AXHd3 and an alpha-L-arabinofuranosidase from Aspergillus niger did not result in a higher maximal release of arabinose than incubation with these enzymes separately.