Cloning of the Ribosomal Protein L41 Gene of Phaffia rhodozyma and Its Use as a Drug Resistance Marker for Transformation

Abstract

The ribosomal protein L41 gene of Phaffia rhodozyma was cloned and used as a dominant selectable marker for cycloheximide resistance in the transformation of P. rhodozyma. Electrotransformation with a plasmid containing a ribosomal DNA fragment as a targeting signal typically yielded 800 to 1,200 transformants/μg of DNA with an integrated copy number of about seven per haploid genome.

Phaffia rhodozyma, a pigmented fermenting yeast, is one of the few microorganisms known to synthesize astaxanthin (3,3′-dihydroxy-β,β′-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 system—higher yields and more stable transformants—for 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 DH5α [endA1 recA1 hsdR17 supE44 thi-1 gyrA96 relA1 lacU169 (φ80 lacZ ΔM15)] was used for construction and propagation of the DNA library and plasmids.

A clone hybridizing with the PCR product, pTPL2 (Fig. 1), was identified, and a 3.5-kb XbaI-SalI fragment was subcloned and sequenced. We also isolated P. rhodozyma L41 cDNA by the method of rapid amplification of cDNA ends (RACE) with 3′-RACE (GIBCO BRL, Gaithersburg, Md.) and 5′-RACE (AmpliFINDER; Clontech, Palo Alto, Calif.) kits. Total RNA was prepared by the method of Elion and Warner (8), and primers corresponding to amino acids 52 to 59 were used for 3′- and 5′-RACE reactions, respectively. The 3′- and 5′-RACE products were sequenced. A putative open reading frame of 1,218 bp interrupted by six introns was found. An additional intron was found in the putative 5′ untranslated mRNA leader. The six nucleotides of the 5′ splice site and three nucleotides of the 3′ splice site of these introns were conserved and were similar to the consensus sequence elements GTPuNGT and PyAG, respectively. The number of introns and their organization in the P. rhodozyma L41 gene were quite different from those of L41 genes in other yeasts (7, 13, 18), where there is only one intron located just downstream of the initiation codon. Phaffia actin introns cannot be spliced in Saccharomyces cerevisiae, so the differences in intron structure are probably significant (24). The deduced amino acid sequence of P. rhodozyma L41 was similar to those from other yeasts (79.2 to 85.8%). All of the cycloheximide (CYH)-resistant yeasts have glutamine at position 56, and CYH-sensitive yeasts have proline at that position.

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.

To clone the ribosomal DNA (rDNA) fragment, two pairs of PCR primers were designed from the known partial rDNA sequence of P. rhodozyma (10, 25): 5′-TCC TAG TAA GCG CAA GTC AT-3′ and 5′-TTC GGC CAA GGA AAG AAA CT-3′ in the 18S region and 5′-AAT CGG ATT ATC CGG AGC TA-3′ and 5′-GCT ATA ACA CAT CCG GAG AT-3′ in the 26S region. Two DNA fragments were obtained by PCR with these two pairs of primers and used as a probe for cloning the rDNA unit. Southern hybridization identified an 8.5-kb HindIII fragment, which was cloned and whose identity was confirmed by partial sequencing. A 730-bp XhoI-XbaI fragment of rDNA which spans the nontranscribed spacer region between 5S and 18S rDNA was subcloned (pTPR4) to construct a plasmid for transformation (Fig. 1). The 3.7-kb XbaI-SalI fragment of pTPL5 containing the L41 gene was treated with the Klenow fragment of DNA polymerase and inserted into the BalI site of pTPR4. The resulting plasmid, pTPLR1, carries the 3.7-kb P. rhodozyma L41 gene conferring CYH resistance and the 730-bp rDNA fragment for targeting into the chromosome (Fig. 1).

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 Ω 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.

To study the fate of the transforming plasmid, genomic DNA was prepared from five CYH-resistant colonies obtained from a transformation with pTPLR1 (linearized with BglI-KpnI). Southern blots of genomic DNA from these transformants were probed with the 2.2-kb SalI fragment of pTPL2 (Fig. 2). Southern hybridization of genomic DNA restricted with SmaI gave rise to a signal at 9.0 kb both in a nontransformant control and in the transformants (Fig. 2A), indicating that this band originated from the endogenous P. rhodozyma L41 gene. A much stronger signal at 4.1 kb also was detected in transformants, but not in the control. This was expected from the restriction map of the transforming plasmid (Fig. 2B). The size and relative intensity of signal at 4.1 kb and the fact that no transformant was obtained with nonlinearized plasmid suggested that multiple copies of the transforming plasmid had been integrated. This band also was detected with an rDNA probe (data not shown). The number of integrated plasmids was estimated to be approximately seven copies per haploid genome by densitometric comparison of the signal intensity of the 4.1-kb band with that of the 9.0-kb control on the blot in a scanning densitometer (Model GS-700 imaging densitometer; Bio-Rad). Copy number did not decrease after a prolonged cultivation in YM medium, indicating that the transforming plasmid was integrated into the chromosome and maintained stably. In another Southern blot with EcoRI digestion, two bands at 5.8 and 2.8 kb were found only in transformants (Fig. 2A). The 5.8-kb band originated from a 3.2-kb rDNA fragment and a 2.6-kb L41 gene fragment, and the 2.8-kb band originated from a 1.7-kb rDNA fragment and a 1.1-kb L41 gene fragment. Integration probably occurs as diagrammed in Fig. 2B.

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.

In most transformants, plasmid integration appeared to occur via homologous recombination into the rDNA locus. There also were a few transformants in which the wild-type chromosomal copy of L41 was replaced with the mutated copy. No transformants were obtained with the intact, circular plasmid, suggesting that no stable autonomously replicating sequence activity existed in the rDNA fragment. Electrotransformation reproducibly resulted in transformation efficiencies of 800 to 1,200 transformants/μg of DNA, which are comparable with that reported in other CYH marker systems (1,400 transformants/μg of DNA for Candida utilis [14]). More careful optimization of transformation conditions in electroporation or in selection of different parts of rDNA could further increase the efficiency. Use of a higher concentration of CYH might result in the selection of transformants with higher copy numbers. The MIC of CYH was not dependent on the density of the plating cells, and false positives were not a problem. The use of an endogenous gene as the selectable marker eliminates the need to introduce foreign DNA sequences for drug resistance into the host organism. The substantially improved transformation efficiency will facilitate procedures for which a larger pool of transformants is required, e.g., complementation cloning of the genes involved in carotenoid biosynthesis and expression cloning of genes by gene dosage effect.

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.