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 beta-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 beta-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 beta-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3'-4'-didehydro-beta-psi-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.

FIG. 1.

The astaxanthin biosynthetic pathway in X. dendrorhous proposed by Andrewes et al. (5). The main carotenoids found after the introduction of additional gene copies of the phytoene desaturase-encoding gene (crtI) in the X. dendrorhous strains CBS 6938 and PR-1-104 (12) are boxed (this study). Roman numbers (I, II, and III) indicate three potential routes for the formation of torulene from neurosporene.

FIG. 2.

Schematic representation of the specific crtYB gene inactivation approach by single (A) and double (B) crossover. Prior to the introduction in X. dendrorhous by electrotransformation, the plasmids pPR16 and pPR19F were linearized with the endonucleases BstXI and EcoRI, respectively. SI, site of insertion (the insert was a blunted EcoRI fragment in a blunted BstXI site) (Table 1).

FIG. 3.

Graphical presentation of the transformation vectors (pPR1 and pPR2TN) and carotenogenic expression vectors (pPR13F, pPR22F, pPR19F, and pPR40F). Depending on the orientation of the carotenogenic expression cassette, the vectors were denominated F (forward) or R (reverse) when the carotenogenic gene was transcribed in the same or opposite direction, respectively, as the G418 marker gene. The Sau3A* (not unique) this site was created by the ligation of BamHI and BglII sites. SI, site of insertion (the insert was a blunted EcoRI fragment in a blunted BstXI site; details are given in Table 1).

FIG. 4.

Autoradiogram of a Southern blot of chromosomal DNA isolated from several transformants of X. dendrorhous strain CBS 6938. Plasmids and chromosomal DNA were digested with the endonucleases indicated at the top of the panels. The blot was hybridized with a DIG-labeled cDNA probe encoding CrtYB. A DNA ladder containing fragments of 10, 8, 6, 5, 4, 3.5, 3 (marked with an asterisk), 2.5, 2, and 1 kb was used as a marker (lanes M). The expected hybridization patterns for transformants obtained after the integration of pPR16 or pPR19F at the crtYB locus are depicted in Fig. 2.

FIG. 5.

Hypothetical representation of the carotenogenic complex in X. dendrorhous (left) and a recombinant strain overexpressing the phytoene desaturase (CrtI)-encoding gene (right). Increased levels of a carotenogenic enzyme might alter the sequence of reactions. In the β-carotene-accumulating strain PR-1-104, the astaxanthin synthetase (Ast) enzyme is inactive or absent (above dotted line). The main carotenoids under each specific condition are indicated in boxes. *, the 3,4-didehydro ends of torulene and HDCO are not substrates for lycopene cyclase (CrtYB) and astaxanthin synthetase (Ast), respectively. The number and stoichiometry of enzymes are speculative and based on the number of different enzymatic steps. CPR, cytochrome P450 reductase.