Efficient generation of recessive traits in diploid sake yeast by targeted gene disruption and loss of heterozygosity

Sake yeast, a diploid Saccharomyces cerevisiae strain, is useful for industry but difficult to genetically engineer because it hardly sporulates. Until now, only a few recessive mutants of sake yeast have been obtained. To solve this problem, we developed the high-efficiency loss of heterozygosity (HELOH) method, which applies a two-step gene disruption. First, a heterozygous disruptant was constructed by gene replacement with URA3, followed by marker recycling on medium containing 5-fluoroorotic acid (5-FOA). Subsequently, spontaneous loss of heterozygosity (LOH) yielding a homozygous disruptant was selected for in a second round of gene integration. During this step, the wild-type allele of the heterozygous disruptant was marked by URA3 integration, and the resulting transformants were cultivated in non-selective medium to induce recombination and then grown on medium with 5-FOA to enrich for mutants that had undergone LOH. Although the frequency with which LOH occurs is extremely low, many homozygous disruptants were obtained with the HELOH method. Thus, we were able to efficiently construct homozygous disruptants of diploid sake yeast without sporulation, and sake yeast strains with multiple auxotrophies and a protease deficiency could be constructed. The HELOH method, therefore, facilitated the utilization of diploid sake yeast for genetic engineering purposes.     

One-step gene disruption by cotransformation to isolate double auxotrophs in Candida albicans

Summary The Candida albicans LEU2 gene was disrupted by substituting lambda DNA for a small deletion within the LEU2 gene. Cotransformation with a selectable URA3 ARS vector was used to introduce a linear fragment containing the disruption into the genome of a C. albicans ura3 deletion mutant. Cotransformants containing the lambda DNA were identified by colony hybridization and the URA3 plasmid was subsequently cured. Leu2 disrupted heterozygotes were detected by Southern hybridization and one disruptant was subsequently treated with UV irradiation. Only one leu2 ura3 mutant (SGY-484) was isolated out of 11,000 mutagenized cells. SGY-484 was transformed to Leu⁺ with either the C. albicans or Saccharomyces cerevisiae LEU2 gene. Southern hybridization analysis revealed that the mutant is not homozygous for the disruption; the leu2 mutation reverts and is most likely a point mutation. Unexpectedly, an ade2 ura3 mutant was isolated from the same mutagenesis.     

Directed mutagenesis in Candida albicans: one-step gene disruption to isolate ura3 mutants.

A method for introducing specific mutations into the diploid Candida albicans by one-step gene disruption and subsequent UV-induced recombination was developed. The cloned C. albicans URA3 gene was disrupted with the C. albicans ADE2 gene, and the linearized DNA was used for transformation of two ade2 mutants, SGY-129 and A81-Pu. Both an insertional inactivation of the URA3 gene and a disruption which results in a 4.0-kilobase deletion were made. Southern hybridization analyses demonstrated that the URA3 gene was disrupted on one of the chromosomal homologs in 15 of the 18 transformants analyzed. These analyses also revealed restriction site dimorphism of EcoRI at the URA3 locus which provides a unique marker to distinguish between chromosomal homologs. This enabled us to show that either homolog could be disrupted and that disrupted transformants of SGY-129 contained more than two copies of the URA3 locus. The A81-Pu transformants heterozygous for the ura3 mutations were rendered homozygous and Ura- by UV-induced recombination. The homozygosity of a deletion mutant and an insertion mutant was confirmed by Southern hybridization. Both mutants were transformed to Ura+ with plasmids containing the URA3 gene and in addition, were resistant to 5-fluoro-orotic acid, a characteristic of Saccharomyces cerevisiae ura3 mutants as well as of orotidine-5'-phosphate decarboxylase mutants of other organisms.     

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.     

Identification and characterization of a novel biotin biosynthesis gene in Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae cells generally cannot synthesize biotin, a vitamin required for many carboxylation reactions. Although sake yeasts, which are used for Japanese sake brewing, are classified as S. cerevisiae, they do not require biotin for their growth. In this study, we identified a novel open reading frame (ORF) in the genome of one strain of sake yeast that we speculated to be involved in biotin synthesis. Homologs of this gene are widely distributed in the genomes of sake yeasts. However, they are not found in many laboratory strains and strains used for wine making and beer brewing. This ORF was named BIO6 because it has 52% identity with BIO3, a biotin biosynthesis gene of a laboratory strain. Further research showed that yeasts without the BIO6 gene are auxotrophic for biotin, whereas yeasts holding the BIO6 gene are prototrophic for biotin. The BIO6 gene was disrupted in strain A364A, which is a laboratory strain with one copy of the BIO6 gene. Although strain A364A is prototrophic for biotin, a BIO6 disrupted mutant was found to be auxotrophic for biotin. The BIO6 disruptant was able to grow in biotin-deficient medium supplemented with 7-keto-8-amino-pelargonic acid (KAPA), while the bio3 disruptant was not able to grow in this medium. These results suggest that Bio6p acts in an unknown step of biotin synthesis before KAPA synthesis. Furthermore, we demonstrated that expression of the BIO6 gene, like that of other biotin synthesis genes, was upregulated by depletion of biotin. We conclude that the BIO6 gene is a novel biotin biosynthesis gene of S. cerevisiae.     

Elevated Growth of Saccharomyces cerevisiae ATH1 Null Mutants on Glucose Is an Artifact of Nonmatching Auxotrophies of Mutant and Reference Strains

Yeast strains disrupted for ATH1, which encodes vacuolar acid trehalase, have been reported to grow to higher cell densities than reference strains. We showed that the increase in cell density is due to the URA3 gene introduced as a part of the disruption and concluded that the misinterpretation is a result of not using a control strain with matching auxotrophic markers.     

Metabolic engineering of Torulopsis glabrata for improved pyruvate production

During pyruvate production, ethanol is produced as a by-product, which both decreases the amount of pyruvate and makes the recovery of pyruvate more difficult. Pyruvate decarboxylase (PDC, EC 4.1.1.1), which degrades pyruvate to acetaldehyde and ultimately to ethanol, is a key enzyme in the pyruvate metabolism of yeast. Therefore, to order to increase the yield of pyruvate in Torulopsis glabrata, targeted PDC-disrupted strains were metabolically engineered. First, T. glabrata ura3 strains that were suitable for genetic transformation were isolated and identified through ethyl methansulfonate mutagenesis, 5-fluoroortic acid media selection, and Sacchramyces cerevisiae URA3 complement. Next, the PDC gene in T. glabrata was specifically disrupted through homologous recombinant with the S. cerevisiae URA3 gene as the selective marker. The PDC activity of the disruptants was about 33% that of the parent strain. Targeted PDC gene disruption in T. glabrata was also confirmed by PCR amplification and sequencing of the PDC gene and its mutants, PDC activity staining, and PDC Western blot. The disruptants displayed higher pyruvate accumulation and less ethanol production. Under basal fermentation conditions (see Section 2), the disruptants accumulated about 20 g/L of pyruvate with 4.6 g/L of ethanol, whereas the parental strain (T. glabrata IFO005) only accumulated 7–8 g/L of pyruvate with 7.4 g/L of ethanol. Under favorable conditions in jar fermentation, the disruptants accumulated 82.2 g/L of pyruvate in 52 h.     

An Acid- and Heat-stable β-Fructofuranosidase from Corticium rolfsii

We reported previously that an ndhB gene disruptant, ΔndhB, had the same phenotype as wild-type tobacco plants under normal growth conditions. Two other groups have reported conflicting phenotypes with each other for ndhCKJ operon disruptants. Here, we generated two transformants in which the ndhCKJ operon was disrupted, and found that new transformants had the same phenotype as ΔndhB. After illumination with visible light, all ndh disruptants had higher levels of steady-state fluorescence than wild-type controls when measured under weak light, suggesting that reduction of the plastoquinone pool in ndh disruptants was greater than that in wild-type controls. The weak light itself could not reduce the plastoquinone much, so the reduction in the plastoquinone in the mutant was due to electron donation from stromal reductants generated during illumination with the strong light. These results supported the hypothesis that NAD(P)H dehydrogenase prevents overreduction in chloroplasts and suggested that chlororespiratory oxidase did not function under low light or in the dark.     

Marker-disruptive gene integration and URA3 recycling for multiple gene manipulation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The introduction of several kinds of genes into the yeast chromosome is a powerful tool in many fields from fundamental study to industrial application. Here, we describe a general strategy for one-step gene integration and a marker recycling method. Forty base pairs of a short sequence derived from a region adjacent to the HIS3 locus were placed between cell surface displaying β-glucosidase (BGL) and URA3 marker genes. HIS3 deletion and BGL–URA3 fragment integration were achieved via a PCR fragment consisting of the BGL–URA3 fragment attached to homology sequences flanked by the HIS3 targeting locus. The obtained his3::URA3 disruptants were plated on a 5-FOA plate to select for the URA3 deletion due to repeated sequences at both sides of URA3 gene. In all selected colonies, BGL genes were integrated at the targeted HIS3 locus and URA3 was completely deleted. In addition, introduced BGL was efficiently expressed, and the transformants fermented cellobiose to ethanol effectively. As our strategy creates next transformation markers continuously together with gene integration, this method can serve as a simple and powerful tool for multiple genetic manipulations in yeast engineering.     

Construction of a Quadruple Auxotrophic Mutant of an Industrial Polyploid Saccharomyces cerevisiae Strain by Using RNA-Guided Cas9 Nuclease

Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries.     

An efficient method for homologous gene reconstitution in Cryptococcus gattii using URA5 auxotrophic marker

Cryptococcus gattii (Cg) is an emerging pathogen of both healthy and immunocompromised patients worldwide. Understanding the molecular genetic basis of virulence and physiology of this pathogen will be critical for defining its pathogenic mechanisms. The purine biosynthetic gene, URA5 encoding orate phosphorybosyltransferase (OPRTase), has been successfully used as a selectable marker for gene disruption by transformation and homologous recombination in Cg. Here, we report the characterization of ura5 auxotrophy and URA5 reversion phenomenon at the molecular, genetic, and structural levels, and use of ura5→URA5 reversion as a tool for reconstitution of gene of interest and auxotrophic marker to their native loci. We identified a single mutation of GG₁₂₈T→GAT with substitution of glycine to aspartic acid at amino acid position 43 resulting in ura5 auxotrophy. The ura5→URA5 reversion on CSM lacking uracil (CSM-U) was found to be a rare phenomenon with a reversion frequency of 0.000002%, and sequence analysis of URA5 from all the reverted strains revealed mutation of GA₁₂₈T→GGT back to its ancestral state. The URA5 allele in the reverted strains was fully functional, as demonstrated by the excellent growth of these strains on medium lacking uracil, as well as by the ability of this allele to efficiently transform ura5 mutant to restore prototrophy. The deduced Cg URA5 protein modeled on the known crystal structures of OPRTase from Salmonella typhimurium (1LH0_A, 1STO) and from Escherichia coli (1ORO_A) indicated that the glycine 43 of Cg URA5 was situated on a conserved loop, and it’s substitution to more globose aspartic acid may have resulted in URA5 inactivation in auxotrophic strain. The advantages of this approach for the generation of a reconstituted strain are (1) that it restores the functionality of the native URA5, (2) that it eliminates an additional biolistic delivery of exogenous URA5, and (3) that it allows easy selection of reconstituted strains with homologous integration. This strategy was successfully used for the generation of Cg can2+CAN2/URA5 homologous reconstituted strains, which grew in ambient air to the wild-type level while can2 mutant exhibited severe growth defect under similar conditions. Srinivas D. Narasipura and Ping Ren contributed equally to this work.     

Development of an efficient genetic manipulation strategy for sequential gene disruption and expression of different heterologous <Emphasis Type="Italic">GFP</Emphasis> genes in <Emphasis Type="Italic">Candida tropicalis</Emphasis>

The diploid yeast Candida tropicalis, which can utilize n-alkane as a carbon and energy source, is an attractive strain for both physiological studies and practical applications. However, it presents some characteristics, such as rare codon usage, difficulty in sequential gene disruption, and inefficiency in foreign gene expression, that hamper strain improvement through genetic engineering. In this work, we present a simple and effective method for sequential gene disruption in C. tropicalis based on the use of an auxotrophic mutant host defective in orotidine monophosphate decarboxylase (URA3). The disruption cassette, which consists of a functional yeast URA3 gene flanked by a 0.3 kb gene disruption auxiliary sequence (gda) direct repeat derived from downstream or upstream of the URA3 gene and of homologous arms of the target gene, was constructed and introduced into the yeast genome by integrative transformation. Stable integrants were isolated by selection for Ura⁺ and identified by PCR and sequencing. The important feature of this construct, which makes it very attractive, is that recombination between the flanking direct gda repeats occurs at a high frequency (10^?8) during mitosis. After excision of the URA3 marker, only one copy of the gda sequence remains at the recombinant locus. Thus, the resulting ura3 strain can be used again to disrupt a second allelic gene in a similar manner. In addition to this effective sequential gene disruption method, a codon-optimized green fluorescent protein-encoding gene (GFP) was functionally expressed in C. tropicalis. Thus, we propose a simple and reliable method to improve C. tropicalis by genetic manipulation.     

A new method for isolation of S-adenosylmethionine (SAM)-accumulating yeast

S-Adenosylmethionine (SAM) is an important metabolite that participates in many reactions as a methyl group donor in all organisms, and has attracted much interest in clinical research because of its potential to improve many diseases, such as depression, liver disease, and osteoarthritis. Because of these potential applications, a more efficient means is needed to produce SAM. Accordingly, we developed a positive selection method to isolate SAM-accumulating yeast in this study. In Saccharomyces cerevisiae, one of the main reactions consuming SAM is thought to be the methylation reaction in the biosynthesis of ergosterol that is catalyzed by Erg6p. Mutants with deficiencies in ergosterol biosynthesis may accumulate SAM as a result of the reduction of SAM consumption in ergosterol biosynthesis. We have applied this method to isolate SAM-accumulating yeasts with nystatin, which has been used to select mutants with deficiencies in ergosterol biosynthesis. SAM-accumulating mutants from S. cerevisiae K-9 and X2180-1A were efficiently isolated through this method. These mutants accumulated 1.7–5.5 times more SAM than their parental strains. NMR and GC-MS analyses suggested that two mutants from K-9 have a mutation in the erg4 gene, and erg4 disruptants from laboratory strains also accumulated more SAM than their parental strains. These results indicate that mutants having mutations in the genes for enzymes that act downstream of Erg6p in ergosterol biosynthesis are effective in accumulating SAM.     

Inactivation of OGG1 increases the incidence of G?·?C→T?·?A transversions in Saccharomyces cerevisiae : evidence for endogenous oxidative damage to DNA in eukaryotic cells

The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. To investigate the biological role of the OGG1 gene, mutants were constructed by partial deletion of the coding sequence and insertion of marker genes, yielding ogg1::TRP1 and ogg1::URA3 mutant strains. The disruption of the OGG1 gene does not compromise the viability of haploid cells, therefore it is not an essential gene. The capacity to repair 8-OxoG has been measured in cell-free extracts of wild-type and ogg1 strains using a 34mer DNA fragment containing a single 8-OxoG residue paired with a cytosine (8-OxoG/C) as a substrate. Cell-free extracts of the wild-type strain efficiently cleave the 8-OxoG-containing strand of the 8-OxoG/C duplex. In contrast, cell-free extracts of the Ogg1-deficient strain have no detectable activity that can cleave the 8-OxoG/C duplex. The biological properties of the ogg1 mutant have also been investigated. The results show that the ogg1 disruptant is not hypersensitive to DNA-damaging agents such as ultraviolet light at 254 nm, hydrogen peroxide or methyl methanesulfonate. However, the ogg1 mutant exhibits a mutator phenotype. When compared to those of a wild-type strain, the frequencies of mutation to canavanine resistance (Can^R) and reversion to Lys⁺ are sevenfold and tenfold higher for the ogg1 mutant strain, respectively. Moreover, using a specific tester system, we show that the Ogg1-deficient strain displays a 50-fold increase in spontaneously occurring G · C→T · A transversions compared to the wild-type strain. The five other base substitution events are not affected by the disruption of the OGG1 gene. These results strongly suggest that endogeneous reactive oxygen species cause DNA damage and that the excision of 8-OxoG catalyzed by the Ogg1 protein contributes to the maintenance of genetic stability in S. cerevisiae. Received: 6 September 1996 / Accepted: 22 October 1996     

Novel and convenient methods for Candida tropicalis gene disruption using a mutated hygromycin B resistance gene

We established a novel and convenient method to construct a ura3 strain (ura3/ura3) of the asporogenous and diploid yeast, Candida tropicalis, that produces dicarboxylic acid. One copy of the URA3 gene was disrupted using a mutated hygromycin B resistance gene (HYG#). The obtained hygromycin-resistant strain was further transformed with a URA3 disruption cassette and selected on a plate containing 5-fluoroorotic acid. The obtained strains were analyzed and the disruption of the gene was confirmed by PCR and Southern blot analysis. The results showed that the strains were obtained in which allelic URA3 genes were simultaneously disrupted. Furthermore, we established a cotransformation method for this gene disruption, using HYG# in C. tropicalis. In order to disrupt the allelic POX4 genes (encoding acyl-CoA oxidase) of dicarboxylic acid-producing strains, the ARS plasmid (which contained HYG#) and a POX4 disruption cassette (which carried the LAC4 gene encoding beta-galactosidase of Kluyveromyces lactis) were simultaneously introduced by transformation. As a result, the allelic POX4 gene was successfully disrupted.     

Characterization of the methyltransferases in the yeast phosphatidylethanolamine methylation pathway by selective gene disruption

PEM1 and PEM2 are structural genes for the yeast phosphatidylethanolamine methylation pathway which mediates the three-step methylation of phosphatidylethanolamine to phosphatidylcholine. Selective disruption of each locus in the yeast genome was performed using the in-vitro-inactivated gene with insertion of yeast LEU2 or HIS3. Complementation test and spore analysis indicated that the disruptants were allelic with our previous mutants that were isolated by chemical mutagenesis and used for the cloning of PEM1 and PEM2. The methyltransferase activities of the disruptants were assayed using their membrane fractions. When the PEM1 locus was disrupted, the activity for the first methylation was greatly decreased but was still detectable, while the activities for the second and third methylations were well retained. The remaining three activities exhibited nearly identical pH optima and apparent Km values for S-adenosyl-L-methionine. The disruptant incorporated radioactivity from L-[methyl-14C]Met into phosphatidylcholine at a low but measurable rate and required choline for optimal growth. When choline was omitted from the culture medium, the phosphatidylcholine content of the cells significantly decreased, but was restored by the addition of N-monomethylethanolamine or choline. When the PEM2 locus was disrupted, the activities for the second and third methylations were totally lost, but that for the first methylation remained. This activity could be distinguished from those remaining in the pem1 disruptant by its different pH optimum and apparent Km for S-adenosyl-L-methionine. When incubated with [methyl-14C]Met, the pem2 disruptant accumulated the radioactivity in phosphatidylmonomethylethanolamine. This disruptant also required choline for optimal growth. In the absence of choline, it accumulated phosphatidylmonomethylethanolamine with a concomitant decrease in phosphatidylcholine and phosphatidylethanolamine. When both loci were disrupted, all phospholipid-methylating activities were lost and cells absolutely required choline for growth. The flux through the pathway became negligible. Thus, the PEM1-encoded methyltransferase was strictly specific to the first step while the PEM2-encoded methyltransferase exhibited a somewhat broader specificity with a preference for the second and third steps of the pathway. These two enzymes accounted for all the activities in the yeast phosphatidylethanolamine methylation pathway.     

Development of an efficient gene targeting system in Colletotrichum higginsianum using a non-homologous end-joining mutant and Agrobacterium tumefaciens-mediated gene transfer

The hemibiotrophic ascomycete Colletotrichum higginsianum is the casual agent of anthracnose disease of cruciferous plants. High efficiency transformation by Agrobacterium tumefaciens-mediated gene transfer has been established for this fungus. However, targeted gene mutagenesis through homologous recombination rarely occurs in C. higginsianum. We have identified and disrupted the C. higginsianum homologue of the human Ku70 gene, ChKU70, which encodes a protein that plays a role in non-homologous end-joining for repair of DNA breaks. chku70 mutants showed a dramatic increase in the frequency of integration of introduced exogenous DNA fragments by homologous recombination without any detectable phenotypic defects. This result demonstrates that the chku70 mutant is an efficient recipient for targeted gene mutagenesis in C. higginsianum. We have also developed a novel approach [named direct repeat recombination-mediated gene targeting (DRGT)] for targeted gene disruption through Agrobacterium tumefaciens-mediated gene transfer. DRGT utilizes homologous recombination between repeated sequences on the T-DNA flanking a partial fragment of the target gene. Our results suggest that DRGT in the chku70 mutant background could be a useful tool for rapid isolation of targeted gene disruptants in C. higginsianum.     

Development of a novel quadruple auxotrophic host transformation system by argB gene disruption using adeA gene and exploiting adenine auxotrophy in Aspergillus oryzae

We previously designed a triple auxotrophic host-vector system in Aspergillus oryzae by isolating red-colored adenine auxotrophic mutants upon UV mutagenesis of a double auxotrophic host (niaD-sC-). In the present study an effort to exploit this system and construct a novel quadruple auxotrophic host was made by disrupting the argB gene involved in arginine biosynthesis. The argB gene-disruption cassette was generated by fusion PCR, which required only two steps of PCR to insert the selectable marker, adeA, into the target argB gene. The chimeric DNA fragment was transformed into the triple auxotrophic strain (niaD-sC-adeA-) and the argB disruptants were obtained with a high rate of efficiency (approximately 40%). The argB disruptants were characterized by normal colony color and reversal of arginine auxotrophy by introduction of the wild-type argB gene. Quadruple auxotrophic strains (niaD-sC-DeltaargB adeA- or niaD-sC-DeltaargB adeB-) were subsequently isolated upon UV mutagenesis of the triple auxotrophic strain (niaD-sC-DeltaargB) followed by screening of red-colored colonies for adenine auxotrophy. The results obtained showed that the adeA gene served as an efficient selection marker in developing a novel host-vector system with quadruple auxotrophy in A. oryzae, thus providing a powerful tool to breed multiple auxotrophic mutants from a deuteromycete wherein sexual crossing is impossible.     

Elevated growth of Saccharomyces cerevisiae ATH1 null mutants on glucose is an artifact of nonmatching auxotrophies of mutant and reference strains

Yeast strains disrupted for ATH1, which encodes vacuolar acid trehalase, have been reported to grow to higher cell densities than reference strains. We showed that the increase in cell density is due to the URA3 gene introduced as a part of the disruption and concluded that the misinterpretation is a result of not using a control strain with matching auxotrophic markers.