Transformation system for an asporogenous methylotrophic yeast, Candida boidinii: cloning of the orotidine-5''-phosphate decarboxylase gene (URA3), isolation of uracil auxotrophic mutants, and use of the mutants for integrative transformation.

An integrative transformation system was established for an asporogenous methylotrophic yeast, Candida boidinii. This system uses a uracil auxotrophic mutant of C. boidinii as the host strain in combination with its URA3 gene as the selectable marker. First, the C. boidinii URA3 gene coding for orotidine-5'-phosphate decarboxylase (ODCase) was cloned by using complementation of the pyrF mutation of Escherichia coli. Next, the host ODCase-negative mutant strains (ura3 strains) were isolated by mutagenesis and selection for 5-fluro-orotic acid (5-FOA) resistance. Five ura3 host strains that exhibited both a low reversion rate and good methylotrophic growth were obtained. All of these strains could be transformed to Ura+ phenotype with a C. boidinii URA3-harboring plasmid linearized within the Candida DNA. The transformants had a stable Ura+ phenotype after nonselective growth for 10 generations. These results and extensive Southern analysis indicated that the linearized plasmid was integrated into the host chromosomal DNA by homologous recombination at the URA3 locus in C. boidinii.     

Identification of the Orotidine-5′-Phosphate Decarboxylase Gene and Development of a Transformation System in the Yeast Saccharomyces exiguus Yp74L-3

To investigate the uracil biosynthetic pathway of the yeast Saccharomyces exiguus Yp74L-3, uracil auxotrophic mutants were isolated. Using conventional genetic techniques, four mutant genes concerned in uracil biosynthesis were identified and denoted as ura1, ura2, ura3, and ura4. Mutations in the URA3 and URA4 genes were specifically selected with 5-fluoroorotic acid (5-FOA). Vector plasmids containing the URA3 gene and an autonomously replicating sequence (ARS) of S. cerevisiae produced sufficient amounts of Ura⁺ transformants from the ura4 mutant of S. exiguus. This fact indicates that the S. exiguus URA4 gene encodes orotidine-5′-phosphate decarboxylase (OMP decarboxylase) and demonstrates that vector plasmids for S. cerevisiae are also usable in S. exiguus.     

Construction of Uracil and Tryptophan Auxotrophic Mutants from Sake Yeasts by Disruption of URA3 and TRP1 Genes

Uracil auxotrophic mutants were constructed from the sake yeasts K-701 and K-901 by successive URA3 gene disruption. First, as sake yeast is diploid, one URA3 gene was disrupted with pURA38 (AURA3 SMR1) and the heterozygous disruptant was isolated on an SM (sulfometuron methyl) plate. The other URA3 gene was disrupted with pURA36 (Δ URA3) and homozygous URA3 disruptants were isolated on FOA (5-fluoro-orotic acid) plate on which only ura3 mutants can grow. Direct URA3 gene disruption with pURA36 (Δ URA3) was also done and the uracil auxotrophic mutant was isolated. Four types of URA3 disruptants were isolated, two of which had no bacterial DNA.

A tryptophan auxotrophic mutant was constructed from one of the URA3 disruptant using pTRP14 (Δ TRP1 URA3) by gene disruption. This TRP1 disruptant was also lacking bacterial DNA.

Laboratory scale sake brewing using the auxotrophic mutants showed that these strains are very useful as recipient strains for molecular breeding of sake yeasts.     

Rare Homologous Gene Targeting in Histoplasma capsulatum: Disruption of the URA5Hc Gene by Allelic Replacement

URA5 genes encode orotidine-5′-monophosphate pyrophosphorylase (OMPpase), an enzyme involved in pyrimidine biosynthesis. We cloned the Histoplasma capsulatum URA5 gene (URA5_Hc) by using a probe generated by PCR with inosine-rich primers based on relatively conserved sequences in OMPpases from other organisms. Transformation with this gene restored uracil prototrophy and OMPpase activity to UV-mutagenized ura5 strains of H. capsulatum. We attempted to target the genomic URA5 locus in this haploid organism to demonstrate homologous allelic replacement with transforming DNA, which has not been previously done in H. capsulatum and has been challenging in some other pathogenic fungi. Several strategies commonly used in Saccharomyces cerevisiae and other eukaryotes were unsuccessful, due to the frequent occurrence of ectopic integration, linear plasmid formation, and spontaneous resistance to 5-fluoroorotic acid, which is a selective agent for URA5 gene inactivation. Recent development of an efficient electrotransformation system and of a second selectable marker (hph, conferring hygromycin B resistance) for this fungus enabled us to achieve allelic replacement by using transformation with an insertionally inactivated Δura5_Hc::hph plasmid, followed by dual selection with hygromycin B and 5-fluoroorotic acid, or by screening hygromycin B-resistant transformants for uracil auxotrophy. The relative frequency of homologous gene targeting was approximately one allelic replacement event per thousand transformants. This work demonstrates the feasibility but also the potential challenge of gene disruption in this organism. To our knowledge, it represents the first example of experimentally directed allelic replacement in H. capsulatum, or in any dimorphic systemic fungal pathogen of humans.     

Development of an integrative DNA transformation system for the yeast Hansenula anomala

A host-vector system for the yeast Hansenula anomala was developed. The system was based on an auxotrophic mutant host of H. anomala which was defective in orotidine-5′-phosphate decarboxylase (ODCase) activity. The H. anomala ODCase-negative mutant strains (ura3 strains) were isolated based on 5-fluoroorotic acid (5-FOA) resistance. A plasmid vector containing the H. anomala URA3 gene was used for transformation. Using this plasmid, all of the H. anomala ura3 strains tested could be transformed to Ura⁺ phenotypes. In all of Ura⁺ transformants, the introduced plasmid was integrated into the chromosomal URA3 locus by homologous recombination. The Ura⁺ phenotype of the transformants was stably maintained after nonselective growth.     

Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress

Very high ethanol tolerance is a distinctive trait of the yeast Saccharomyces cerevisiae with notable ecological and industrial importance. Although many genes have been shown to be required for moderate ethanol tolerance (i.e., 6 to 12%) in laboratory strains, little is known of the much higher ethanol tolerance (i.e., 16 to 20%) in natural and industrial strains. We have analyzed the genetic basis of very high ethanol tolerance in a Brazilian bioethanol production strain by genetic mapping with laboratory strains containing artificially inserted oligonucleotide markers. The first locus contained the ura3Δ0 mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage for LYS2, LEU2, HIS3, and MET15. Tolerance to only very high ethanol concentrations was reduced by auxotrophies, while the effect was reversed at lower concentrations. Evaluation of other stress conditions showed that the link with auxotrophy is dependent on the type of stress and the type of auxotrophy. When the concentration of the auxotrophic nutrient is close to that limiting growth, more stress factors can inhibit growth of an auxotrophic strain. We show that very high ethanol concentrations inhibit the uptake of leucine more than that of uracil, but the 500-fold-lower uracil uptake activity may explain the strong linkage between uracil auxotrophy and ethanol sensitivity compared to leucine auxotrophy. Since very high concentrations of ethanol inhibit the uptake of auxotrophic nutrients, the active uptake of scarce nutrients may be a major limiting factor for growth under conditions of ethanol stress.     

Establishment of an overall transformation system for an oil-producing filamentous fungus,Mortierella alpina 1S-4

Oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. To determine its physiological properties and to clarify the biosynthetic pathways for polyunsaturated fatty acids, a transformation system for this fungus was established using a derivative of it, i.e., a ura5^– mutant lacking orotate phosphoribosyl transferase (OPRTase, EC.2.4.2.10) activity. Transformation with a vector containing the homologous ura5 gene as a marker was successfully performed using microprojectile bombardment, other methods frequently used for transformation, such as the protoplasting, lithium acetate, or electroporation methods, not giving satisfactory results. As a result, two types of transformants were obtained: a few stable transformants overexpressing the ura5 gene, and many unstable transformants showing OPRTase activity comparable to that of the wild-type strain. The results of quantitative PCR indicated that the stable transformants could retain the ura5 genes originating from the transformation vector regardless of the culture conditions. On the other hand, unstable transformants easily lost the marker gene under uracil-containing conditions, as expected. In this paper, we report that an overall transformation system for this fungus was successfully established, and propose how to select useful transformants as experimental and industrial strains.     

Cloning and sequencing of the ura3 and ura5 genes, and isolation and characterization of uracil auxotrophs of the fungus Mortierella alpina 1S-4

The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. In order to prepare host strains for a transformation system for this fungus, six uracil auxotrophs were obtained by means of random mutation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). When the activities of orotate phosphoribosyl transferase (OPRTase, EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (OMPdecase, EC 4.1.1.23) were examined in the mutants and wild strain, OPRTase activity was found to be completely absent in all mutants, on the other hand, OMPdecase activity was intact. The genomic DNA and cDNA of the ura5 gene encoding OPRTase and the ura3 gene encoding OMPdecase were cloned and sequenced. The Ura5p deduced amino acid sequence of this fungus showed highest similarity to that of Vibrio cholerae classed among prokaryote. Furthermore, the mutational points in the ura5 genes of two selected mutants were identified; a base-replacement and a base-insertion.     

Establishment of a selection marker recycling system for sequential transformation of the plant-pathogenic fungus Colletotrichum orbiculare

Genome sequencing of pathogenic fungi has revealed the presence of various effectors that aid pathogen invasion by the manipulation of plant immunity. Effectors are often individually dispensable because of duplication and functional redundancy as a result of the arms race between host plants and pathogens. To study effectors that have functional redundancy, multiple gene disruption is often required. However, the number of selection markers that can be used for gene targeting is limited. Here, we established a marker recycling system that allows the use of the same selection marker in successive transformations in the model fungal pathogen Colletotrichum orbiculare, a causal agent of anthracnose disease in plants belonging to the Cucurbitaceae. We identified two C. orbiculare homologues of yeast URA3/pyrG, designated as URA3A and URA3B, which can be used as selection markers on medium with no uridine. The gene can then be removed from the genome via homologous recombination when the fungus is grown in the presence of 5-fluoroorotic acid (5-FOA), a chemical that is converted into a toxin by URA3 activity. The ura3a/b double mutants showed auxotrophy for uridine and insensitivity to 5-FOA. Using the ura3a/b mutants, transformation with the URA3B marker and its removal were successfully applied to disrupt the virulence-related gene, PKS1. The pks1 mutants showed a reduction in virulence, demonstrating that the method can be used to study virulence-related genes in C. orbiculare. The establishment of a URA3-based marker recycling system in plant-pathogenic fungi enables the genetic analysis of multiple genes that have redundant functions, including effector genes.     

Efficient homologous and illegitimate recombination in the opportunistic yeast pathogen Candida glabrata

The opportunistic pathogen Candida glabrata causes significant disease in humans. To develop genetic tools to investigate the pathogenicity of this organism, we have constructed ura3 and his3 auxotrophic strains by deleting the relevant coding regions in a C. glabrata clinical isolate. Linearized plasmids carrying a Saccharomyces cerevisiae URA3 gene efficiently transformed the ura3 auxotroph to prototrophy. Homologous recombination events were observed when the linearized plasmid carried short terminal regions homologous with the chromosome. In contrast, in the absence of any chromosomal homology, the plasmid integrated by illegitimate recombination into random sites in the genome. Sequence analysis of the target sites revealed that for the majority of illegitimate transformants there was no microhomology with the integration site. Approximately 0.25% of the insertions resulted in amino acid auxotrophy, suggesting that insertion was random at a gross level. Sequence analysis suggested that illegitimate recombination is nonrandom at the single-gene level and that the integrating plasmid has a preference for inserting into noncoding regions of the genome. Analysis of the relative numbers of homologous and illegitimate recombination events suggests that C. glabrata possesses efficient systems for both homologous and nonhomologous recombination.     

Transformation by complementation of a uracil auxotroph of the hyper lignin-degrading basidiomycete Phanerochaete sordida YK-624

Phanerochaete sordida YK-624 is a hyper lignin-degrading basidiomycete possessing greater ligninolytic selectivity than either P. chrysosporium or Trametes versicolor. To construct a gene transformation system for P. sordida YK-624, uracil auxotrophic mutants were generated using a combination of ultraviolet (UV) radiation and 5-fluoroorotate resistance as a selection scheme. An uracil auxotrophic strain (UV-64) was transformed into a uracil prototroph using the marker plasmid pPsURA5 containing the orotate phosphoribosyltransferase gene from P. sordida YK-624. This system generated approximately 50 stable transformants using 2 × 10⁷ protoplasts. Southern blot analysis demonstrated that the transformed pPsURA5 was ectopically integrated into the chromosomal DNA of all transformants. The enhanced green fluorescent protein (EGFP) gene was also introduced into UV-64. The transformed EGFP was expressed in the co-transformants driven by P. sordida glyceraldehyde-3-phosphate dehydrogenase gene promoter and terminator regions.     

Development of a transformation system for gene knock-out in the flavinogenic yeast Pichia guilliermondii

Pichia guilliermondii is a representative of a yeast species, all of which over-synthesize riboflavin in response to iron deprivation. Molecular genetic studies in this yeast species have been hampered by a lack of strain-specific tools for gene manipulation. Stable P. guilliermondii ura3 mutants were selected on the basis of 5'-fluoroorotic acid resistance. Plasmid carrying Saccharomyces cerevisiae URA3 gene transformed the mutant strains to prototrophy with a low efficiency. Substitution of a single leucine codon CUG by another leucine codon CUC in the URA3 gene increased the efficiency of transformation 100 fold. Deletion cassettes for the RIB1 and RIB7 genes, coding for GTP cyclohydrolase and riboflavin synthase, respectively, were constructed using the modified URA3 gene and subsequently introduced into a P. guilliermondii ura3 strain. Site-specific integrants were identified by selection for the Rib(-) Ura(+) phenotype and confirmed by PCR analysis. Transformation of the P. guilliermondii ura3 strain was performed using electroporation, spheroplasting or lithium acetate treatment. Only the lithium acetate transformation procedure provided selection of uracil prototrophic, riboflavin deficient recombinant strains. Depending on the type of cassette, efficiency of site-specific integration was 0.1% and 3-12% in the case of the RIB1 and RIB7 genes, respectively. We suggest that the presence of the ARS element adjacent to the 3' end of the RIB1 gene significantly reduced the frequency of homologous recombination. Efficient gene deletion in P. guilliermondii can be achieved using the modified URA3 gene of S. cerevisiae flanked by 0.8-0.9 kb sequences homologous to the target gene.     

Development of an integrative DNA transformation system for the yeast Candida tropicalis.

We developed the alkane and fatty-acid utilizing yeast Candida tropicalis as a host for DNA transformations. The system is based on an auxotrophic mutant host of C. tropicalis which is defective in orotidine monophosphate decarboxylase (ura3). The ura3 host was isolated by mutagenesis and a double-selection procedure that combined nystatin enrichment selection and 5-fluoro-orotic acid resistance selection. As a selectable marker, we isolated and characterized the C. tropicalis URA3 gene. Plasmid vectors that contained the C. tropicalis URA3 gene transformed the C. tropicalis mutant host at a frequency of 10(3) to 10(4) transformants per micrograms of plasmid DNA. Vectors that contained the Saccharomyces cerevisiae URA3 gene could not transform C. tropicalis. DNA transfer was accomplished by modified versions of either spheroplast generation (CaCl2-polyethylene glycol)-fusion or cation (LiCl) procedures developed for S. cerevisiae. Plasmid vectors that had been cut within the C. tropicalis URA3 fragment integrated by homologous recombination at the URA3 locus.     

Sequence analysis of the DdPYR5-6gene coding for UMP synthase in Dictyostelium discoideum and comparison with orotate phosphoribosyl transferases and OMP decarboxylases

Summary A Dictyostelium discoideum DNA fragment that complements the ura3 and the ura5 mutants of Saccharomyces cerevisiae has been sequenced. It contains an open reading frame of 478 codons capable of encoding a polypeptide of molecular weight 52475. This gene, named DdPYR5-6, encodes a bifunctional protein composed of the orotate phosphoribosyl transferase (OPRTase) and the orotidine-5-phosphate decarboxylase (OMPdecase) domains described for UMP synthase in mammals. The existence of separate domains for the two activities was suspected because deletion of the N-terminal coding segment of the gene eliminated the ura5 but not the ura3 complementing activity. We have now confirmed that the two parts of the open reading frame share homology with known OPRTase and OMPdecase sequences. Several blocks of sequence are conserved among OPRTase from bacteria, fungi and slime mold and one of them corresponds to the consensus sequence for phosphoribosylbinding sites. The OMPdecase domain shows extensive similarity with the yeast and Neurospora crassa enzymes, suggesting that they have evolved from an ancestral gene which was fused to the OPRTase gene in D. discoideum. It is less related to the bacterial enzyme but all these sequences present conserved blocks of homology which could identify the active site. The codon usage is strongly biased in a manner similar to that found for other D. discoideum genes. The flanking DNA contains homopolymers of A and T and alternating sequences that are characteristic of the gene organization in D. discoideum.     

Growth inhibition of Saccharomyces cerevisiae by the immunosuppressant leflunomide is due to the inhibition of uracil uptake via Fur4p

The immunosuppressant leflunomide inhibits cytokine-stimulated proliferation of lymphoid cells in vitro and also inhibits the growth of the eukaryotic microorganism Saccharomyces cerevisiae. To elucidate the molecular mechanism of action of the drug, two yeast genes which suppress the anti-proliferative effect when present in multiple copies were cloned and designated MLF1 and MLF2 for multicopy suppressor of leflunomide sensitivity. DNA sequencing analysis revealed that the MLF1 gene is identical to the FUR4 gene, which encodes a uracil permease and functions to import uracil efficiently. The MLF2 was found to be identical to the URA3 gene. Excess exogenous uracil also overcomes the anti-proliferative effect of leflunomide on yeast cells. Uracil prototrophy also conferred resistance to leflunomide. Uracil uptake was inhibited by leflunomide. Thus, the growth inhibition by leflunomide seen in a S. cerevisiae ura3 auxotroph is due to the inhibition of the entry of exogenous uracil via the Fur4 uracil permease. Received: 7 May 1998 / Accepted: 16 July 1998     

Mitotic Recombination and Genetic Changes in Saccharomyces cerevisiae during Wine Fermentation

Natural strains of Saccharomyces cerevisiae are prototrophic homothallic yeasts that sporulate poorly, are often heterozygous, and may be aneuploid. This genomic constitution may confer selective advantages in some environments. Different mechanisms of recombination, such as meiosis or mitotic rearrangement of chromosomes, have been proposed for wine strains. We studied the stability of the URA3 locus of a URA3/ura3 wine yeast in consecutive grape must fermentations. ura3/ura3 homozygotes were detected at a rate of 1 × 10⁻⁵ to 3 × 10⁻⁵ per generation, and mitotic rearrangements for chromosomes VIII and XII appeared after 30 mitotic divisions. We used the karyotype as a meiotic marker and determined that sporulation was not involved in this process. Thus, we propose a hypothesis for the genome changes in wine yeasts during vinification. This putative mechanism involves mitotic recombination between homologous sequences and does not necessarily imply meiosis.     

Over-expressing <Emphasis Type="Italic">GLT1</Emphasis> in a <Emphasis Type="Italic">gpd2</Emphasis>Δ mutant of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> to improve ethanol production

We constructed two recombinant strains of Saccharomyces cerevisiae in which the GPD2 gene was deleted using a one-step gene replacement method to minimize formation of glycerol and improve ethanol production. In addition, we also over-expressed the GLT1 gene by a two-step gene replacement method to overcome the redox-imbalancing problem in the genetically modified strains. The result of anaerobic batch fermentations showed that the rate of growth and glucose consumption of the KAM-5 (MATα ura3 gpd2Δ::RPT) strain were slower than the original strain, and the KAM-13 (MATα ura3 gpd2Δ::RPT P _PGK -GLT1) strain, however, was indistinguishable compared to the original strain using the same criteria, as analyzed. On the other hand, when compared to the original strain, there were 32 and 38% reduction in glycerol formation for KAM-5 and KAM-13, respectively. Ethanol production increased by 8.6% for KAM-5 and 13.4% for KAM-13. Dramatic reduction in acetate and pyruvic acid was also observed in both mutants compared to the original strains. Although gene GPD2 is responsible for the glycerol synthesis, the mutant KAM-13, in which glycerol formation was substantially reduced, was able to cope and maintain osmoregulation and redox balance and have increased ethanol production under anaerobic fermentations. The result verified the proposed concept of increasing ethanol production in S. cerevisiae by genetic engineering of glycerol synthesis and over-expressing the GLT1 gene along with reconstituted nicotinamide adenine dinucleotide metabolism.     

Spontaneous Auxotrophic and Pigmented Mutants Occurring at High Frequency in Bacillus pumilus NRRL B-3275

Broth cultures of Bacillus pumilus NRRL B-3275 (BpB1) grown at 25, 30, or 37 C contain 1 to 2% spontaneous auxotrophic mutants in both the exponential and stationary phases of growth. Of 70 such mutants isolated from cultures grown at 37 C, approximately two-thirds reverted at such a high frequency as to preclude their study. Of the remaining 22 mutants, 18 required a single amino acid, 1 required adenine, and 1 required uracil. Two of the auxotrophs each required two unrelated amino acids resulting from two independent mutations. All of the mutations reverted spontaneously. Enhanced reversion of approximately one-third of the mutations was obtained with nitrosoguanidine, ethyl methane sulfonate, or diethyl sulfate, or with more than one of these mutagens. The reversion of one mutation was enhanced by 2-aminopurine. The reversion of the remaining mutations was not enhanced by the above mutagens, nor by mutagens known to induce (and revert) frameshift mutations in other bacterial systems. Nine of 10 mutants examined did not show a selective growth advantage over the parents. All but three of the mutations could be linked by PBS1 transduction to one of the previously described auxotrophic markers in strain BpB1. No evidence was obtained for clustering of the mutations on the BpB1 genome. Six of the mutations conferred a requirement for serine. One linked by transduction to trp-2, three linked to argA1, and two (ser-2, -3) linked to argO1. Pigmented mutants (containing a carotenoid-like pigment), which occur spontaneously in BpB1 cultures at a frequency on the order of 1 to 5 mutants per 10(4) cells, link by transduction to ser-2, -3. Spontaneous mutants of strain BpB1 resistant to rifampin, streptomycin, erythromycin, 5-fluorouracil, or 5-methyltryptophan occur at a frequency similar to that of strains of B. pumilus which do not exhibit a high rate of spontaneous mutation to auxotrophy. It is suggested that certain sites or regions of the BpB1 genome exhibit a high rate of spontaneous mutation.