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Phaffia rhodozyma, a pigmented fermenting yeast, is one of the few microorganisms known to synthesize astaxanthin (3,3'-dihydroxy-,'-carotene-4,4'-dione) (4, 12, 16), a carotenoid pigment widely distributed in marine environments and an important constituent of aquacultural feeds for salmonids (11). Astaxanthin's antioxidant properties (3, 20) and its potential role in the prevention of degenerative diseases (17) have led to an increasing interest in using P. rhodozyma to produce astaxanthin. Although P. rhodozyma is a promising microbial source of astaxanthin, studies of this yeast have focused on physiology (11) and selection of overproducing mutants (2, 6, 15). Little is known about the genetics of P. rhodozyma; the ploidy and the sexual cycle have only recently been described (5, 9). In a flow cytometry study, Calo-Mata and Johnson (5) found that no strains were haploid and that most were polyploids. The perfect state of P. rhodozyma has been found, and a pedogamic sexual process of conjugation has been described recently (9). It is extremely difficult to obtain stable, nonreverting auxotrophic mutants for most strains of P. rhodozyma. The absence of suitable genetically marked strains has hindered the development of techniques for molecular genetic manipulation of P. rhodozyma. Recently, there was reported a transformation system of P. rhodozyma using a bacterial kanamycin resistance gene, which confers G418 resistance on yeasts, under the control of either the bacterial promoter (1) or the P. rhodozyma actin promoter (23). These methods have low transformation efficiencies (1 to 10 transformants per µg of DNA) and are poorly reproducible, which may be attributable to the promoters used. Our objective was to develop an improved transformation systemhigher yields and more stable transformantsfor P. rhodozyma. We selected L41, a component of the large ribosomal subunit, for use as a dominant selectable marker that can be expressed with the native transcriptional and translational machinery.

Cloning and sequencing of the L41 protein gene of P. rhodozyma. PCR was performed with degenerate primers which were based on the conserved regions of other known L41 genes (13). Genomic DNA of P. rhodozyma ATCC 24230 (16) was prepared from cells grown at 20 to 23°C in YM broth (Difco, Detroit, Mich.) as described by Sherman et al. (21) and used for a template for PCR. PCR was performed with AmpliTaq DNA polymerase (Perkin-Elmer Cetus, Foster City, Calif.) for 30 cycles with 30 s of denaturation at 94°C, 30 s of annealing at 50°C, and 30 s of extension at 72°C with primers CYH1 (5'-CGC GTA GTT AAY GTN CCN AAR AC-3') and CYH3 (5'-CCC GGG TYT TGG CYT TYT TRT GRA A-3'). PCR generated a single 700-bp fragment which was larger than the expected size (200 bp) of other yeast L41 genes containing one intron (13, 14, 18). The 700-bp PCR fragment was cloned into pT7 Blue plasmid (Novagen, Madison, Wis.) and sequenced on an automatic DNA sequencer (ABI Model 373A; Applied Biosystems, Foster City, Calif.). To clone a full-length L41 gene, the PCR product was labeled with digoxigenin with a DIG labeling kit (Boehringer Mannheim, Mannheim, Germany) and used as a probe in Southern blot analysis of P. rhodozyma chromosomal DNA. Southern hybridization was performed as described in the work of Sambrook et al. (19) in a solution containing 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% (wt/vol) N-lauroylsarcosine, 0.02% (wt/vol) sodium dodecyl sulfate, 5% (wt/vol) blocking reagent, and 50% (vol/vol) formamide at 42°C. A strong hybridization signal was observed from an 8-kb XbaI fragment, and the XbaI fragments of 7 to 9 kb were isolated and ligated into pBluescript (Stratagene, La Jolla, Calif.) to make a minilibrary. Escherichia coli DH5 [endA1 recA1 hsdR17 supE44 thi-1 gyrA96 relA1 lacU169 (80 lacZ M15)] was used for construction and propagation of the DNA library and plasmids.

View larger version (38K): [in this window] [in a new window]   FIG. 1.   Construction of pTPLR1 carrying a CYH resistance marker and an rDNA fragment. Numbers in parentheses are the sizes of inserts. The blank boxes designate a DNA fragment containing the P. rhodozyma L41 gene, the grey boxes indicate the P. rhodozyma rDNA fragment, and thin lines indicate the pBluescript SK(+) sequence. The exons of the L41 gene are designated by black boxes. pTPR4 was derived from a plasmid which contains an 8.5-kb rDNA fragment of P. rhodozyma. The nucleotide and amino acid sequences are shown to illustrate the in vitro mutagenesis for change of Pro56 to Gln56 in L41. Restriction site abbreviations: Ba, BalI; Bg, BglI; C, ClaI; E, EcoRI; H, HindIII; Kp, KpnI; S, SalI; Sm, SmaI; X, XbaI; Xh, XhoI.

Construction of plasmids for transformation. P. rhodozyma was sensitive to CYH (MIC, ~6 µg of CYH/ml). A 2.2-kb-SalI fragment containing the L41 coding region (Fig. 1) was subjected to site-directed mutagenesis to convert the proline residue at position 56 to glutamine. Mutagenesis was carried out with the QuikChange in vitro mutagenesis kit (Stratagene) as described in the manufacturer's instructions with complementary mutagenic primers corresponding to amino acids 52 to 59, 5'-GTG AAA AAC TTG CTT GGT CTG ACC-3' and 5'-GGT CAG ACC AAG CAA GTT TTT CAC-3' (mutated codons are underlined) (Fig. 1). The 2.2-kb SalI fragment was replaced with the mutated fragment, and the 3.7-kb XbaI-SalI fragment containing the promoter and the coding region of the L41 gene was used for the construction of the plasmid for transformation.

Transformations and Southern analysis of transformants. We used a transformation protocol similar to that developed by Varma et al. for electrotransformation of Cryptococcus neoformans (22). Cells from a log-phase culture in 100 ml of YM medium were harvested, washed twice with equal volumes of electroporation buffer (270 mM sucrose, 10 mM Tris, 1 mM MgCl₂ [pH 8.0]) containing 1 mM dithiothreitol, and resuspended in electroporation buffer without dithiothreitol. Plasmid pTPLR1 (200 ng) was linearized with SmaI or BglI-KpnI, mixed with a 50-µl aliquot (approximately 2 × 10⁷ cells) of the cell suspension, and transferred to a cuvette (0.2-cm electrode gap; Bio-Rad, Hercules, Calif.). For electroporation (Gene Pulser II; Bio-Rad), an electric pulse of 0.8 kV was delivered and internal resistance of 600  was set with a capacitance of 50 µF, generating pulse lengths of 18 to 20 ms. The electroporated cells were resuspended in 1 ml of YM medium and transferred to a test tube for incubation. After being shaken for 14 to 16 h at 23°C, cells were spread on YM agar medium containing 10 µg of CYH per ml and incubated at 23°C for 4 to 5 days. Approximately 30% of cells survived, and transformation efficiencies of 800 to 1,200 transformants per µg of DNA could be routinely obtained with pTPLR1 linearized either by SmaI or by BglI-KpnI. Postincubation of electroporated cells for 14 to 16 h was sufficient for the expression of the CYH resistance gene. Postincubation for less than 10 h yielded virtually no transformants, and postincubation for longer than 24 h did not increase the transformation efficiency. No transformants were obtained with intact, nonlinearized plasmid.

View larger version (58K): [in this window] [in a new window]   FIG. 2.   Southern analysis of transformants obtained with pTPLR1 linearized with BglI-KpnI. (A) Genomic DNA of five CYH-resistant colonies was digested with SmaI or EcoRI and probed with the 2.2-kb SalI fragment of pTPL2, which contains the P. rhodozyma L41 gene. Lanes C are the nontransformant control. (B) Schematic presentation of the mode of integration of the transforming DNA into the chromosome. Restriction site abbreviations are the same as in Fig. 1.

Nucleotide sequence accession number. The nucleotide sequence of the L41 gene has been deposited in GenBank under accession no. AF004672. The rDNA sequence fragment has been deposited in GenBank under accession no. AF016256.