Isolation and sequence analysis of the orotidine-5'-phosphate decarboxylase gene (URA3) of Candida utilis. Comparison with the OMP decarboxylase gene family

The URA3 gene of Candida utilis encoding orotidine-5′-phosphate decarboxylase enzyme was isolated by complementation in Escherichia coli pyrF mutation. The deduced amino-acid sequence is highly similar to that of the Ura3 proteins from other yeast and fungal species. An extensive analysis of the family of orotidine-5′-phosphate decarboxylase is shown. The URA3 gene of C. utilis was able to complement functionally the ura3 mutation of Saccharomyces cerevisiae. The sequence presented here has been deposited in the EMBL data library under Accession Number Y12660. © 1998 John Wiley & Sons, Ltd.     

Cloning of orotidine-5′-phosphate decarboxylase (URA3) gene from sourdough yeast Candida milleri CBS 8195

The nucleotide sequence of the URA3 gene encoding orotidine-5′-phosphate decarboxylase (OMPDCase) in sourdough yeast Candida milleri CBS 8195 was determined by degenerate PCR and genome walking. Sequence analysis revealed the presence of an openreading frame of 810 bp, encoding 269 amino acid residue protein with the highest identity to the OMPDCase of the yeast Saccharomyces cerevisiae. Phylogenetic analysis of deduced amino acid sequence revealed that it shares a high degree of identity with other yeast OMPDCases. The cloned URA3 gene successfully complemented the ura3 mutation in S. cerevisiae, indicating that it encodes a functional OMPDCase in C. millieri CBS 8195.     

Construction of a URA3 deletion strain from the allotetraploid bottom-fermenting yeast Saccharomyces pastorianus

The bottom‐fermenting lager yeast Saccharomyces pastorianus has been proposed to be allotetraploid, containing two S. cerevisiae (Sc)‐type and two S. bayanus (Sb)‐type chromosomes. This chromosomal constitution likely explains why recessive mutants of S. pastorianus have not previously been reported. Here we describe the construction of a ura3 deletion strain derived from the lager strain Weihenstephan34/70 by targeted transformation and subsequent loss of heterozygosity (LOH). Initially, deletion constructs of the Sc and Sb types of URA3 were constructed in laboratory yeast strains in which a TDH3p‐hygro allele conferring hygromycin B resistance replaced ScURA3 and a KanMX cassette conferring G‐418 resistance replaced SbURA3. The lager strain was then transformed with these constructs to yield a heterozygous URA3 disruptant (ScURA3⁺/Scura3Δ::TDH3p‐hygro, SbURA3⁺/Sbura3Δ::KanMX), which was plated on 5‐fluoroorotic acid (5‐FOA) plates to generate the desired Ura^– homozygous disruptant (Scura3Δ::TDH3p‐hygro/Scura3Δ::TDH3p‐hygro Sbura3Δ::KanMX/Sbura3Δ::KanMX) through LOH. This ura3 deletion strain was then used to construct a bottom‐fermenting yeast transformant overexpressing ATF1 that encodes an enzyme that produces acetate esters. The ATF1‐overexpressing transformant produced significantly more acetate esters than the parent strain. The constructed ura3? lager strain will be a useful host for constructing strains of relevance to brewing. Copyright © 2012 John Wiley & Sons, Ltd.     

Comparison of expression systems in the yeasts Saccharomyces cerevisiae,Hansenula polymorpha,Klyveromyces lactis,Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica

We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method (Dalbøge and Heldt-Hansen, 1994) in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4·5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1α and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters, Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning. GenBank Accession Numbers: TEF gene promoter sequence: AF054508; RPS7 gene promoter sequence: AF054509. © 1998 John Wiley & Sons, Ltd.     

Designed construction of recombinant DNA at the ura3Δ0 locus in the yeast Saccharomyces cerevisiae

Recombinant DNAs are traditionally constructed using Escherichia coli plasmids. In the yeast Saccharomyces cerevisiae, chromosomal gene targeting is a common technique, implying that the yeast homologous recombination system could be applied for recombinant DNA construction. In an attempt to use a S. cerevisiae chromosome for recombinant DNA construction, we selected the single ura3Δ0 locus as a gene targeting site. By selecting this single locus, repeated recombination using the surrounding URA3 sequences can be performed. The recombination system described here has several advantages over the conventional plasmid system, as it provides a method to confirm the selection of correct recombinants because transformation of the same locus replaces the pre‐existing selection marker, resulting in the loss of the marker in successful recombinations. In addition, the constructed strains can serve as both PCR templates and hosts for preparing subsequent recombinant strains. Using this method, several yeast strains that contained selection markers, promoters, terminators and target genes at the ura3Δ0 locus were successfully generated. The system described here can potentially be applied for the construction of any recombinant DNA without the requirement for manipulations in E. coli. Interestingly, we unexpectedly found that several G/C‐rich sequences used for fusion PCR lowered gene expression when located adjacent to the start codon. Copyright © 2013 John Wiley & Sons, Ltd.     

In vivo evidence for non-universal usage of the codon CUG in Candida maltosa

An alkane-assimilating yeast Candida maltosa had been studied in order to establish systems suitable for biotransformation of hydrophobic compounds. However, functional expression of heterologous genes tested for this purpose had not been successful in several cases. On the other hand, it had been reported that the codon CUG, a universal leucine codon, is read as serine in C. cylindracea. The same altered codon usage had also been suggested by in vitro experiments in some Candida yeasts which are phylogenetically closely related to C. maltosa. In this study we have shown that the failure in functional expression of a heterologous gene is due to the fact that the codon CUG is read as serine in C. maltosa. This conclusion was drawn from the following experimental results: (1) when a cytochrome P450 gene of C. maltosa containing a CTG codon was expressed in C. maltosa, the corresponding amino acid was found to be serine, and not leucine; (2) a tRNA gene with an almost identical structure to that of the tRNA ^SerCAG gene of C. albicans could be isolated from the genome of C. maltosa; (3) the Saccharomyces cerevisiae URA3 gene, which has one CTG codon, could not complement the ura3 mutation of C. maltosa as itself, but when the CTG codon was changed to another leucine codon, CTC, the mutated gene could complement the ura3 mutation. The last result is the first example of succeeding in functional expression of a heterologous gene in Candida species having an altered codon usage by changing the CTG codon in the gene to another codon. The nucleotide sequence datum reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the Accession Number D26074.     

Distribution of the actin cytoskeleton during the cell cycle of Yarrowia lipolytica and the visualization of the tubulin cytoskeleton by immunofluorescence

Actin distribution was examined during the cell cycle of the dimorphic yeast Yarrowia lipolytica, showing the correlation between bud growth, nuclear migration and rearrangement of the actin cytoskeleton. The results correspond with observations made in cells of Saccharomyces cerevisiae, S. uvarum and Candida albicans. Localization of actin was also determined in hyphal cells, where actin is stained predominantly in the tip and also at the septum of hyphae. The standard methods used for tubulin immunostaining in S. cerevisiae and C. albicans cells were adapted for application in Y. lipolytica. Copyright © 1999 John Wiley & Sons, Ltd.     

Isolation and sequence analysis of the gene URA3 encoding the orotidine-5'-phosphate decarboxylase from Candida glycerinogenes WL2002-5, an industrial glycerol producer

The URA3 gene of Candida glycerinogenes WL2002-5, an industrial glycerol producer encoding orotidine-5'-phosphate decarboxylase enzyme, was isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis revealed the presence of an open reading frame (ORF) of 786 bp, encoding a 262 amino acid protein, which shares 71.65% amino acid sequence similarity to the S. cerevisiae URA3 protein. Furthermore, the cloned ORF fully complemented the ura3 mutation of S. cerevisiae, confirming that it encodes for the C. glycerinogenes Ura3 (CgUra3) protein.     

Targeted gene replacement at the URA3 locus of the basidiomycetous yeast Pseudozyma antarctica and its transformation using lithium acetate treatment

The basidiomycetous yeast Pseudozyma antarctica is a remarkable producer of industrially valuable enzymes and extracellular glycolipids. In this study, we developed a method for targeted gene replacement in P. antarctica. In addition, transformation conditions were optimized using lithium acetate, single‐stranded carrier DNA and polyethylene glycol (lithium acetate treatment), generally used for ascomycetous yeast transformation. In the rice‐derived P. antarctica strain GB‐4(0), PaURA3, a homologue of the Saccharomyces cerevisiae orotidine‐5′‐phosphate decarboxylase gene (URA3), was selected as the target locus. A disruption cassette was constructed by linking the nouseothricine resistance gene (natMX4) to homologous DNA fragments of PaURA3, then electroporated into the strain GB‐4(0). We obtained strain PGB015 as one of the PaURA3 disruptants (Paura3Δ::natMX4). Then the PCR‐amplified PaURA3 fragment was introduced into PGB015, and growth of transformant colonies but not background colonies was observed on selective media lacking uracil. The complementation of uracil‐auxotrophy in PGB015 by introduction of PaURA3 was also performed using lithium acetate treatment, which resulted in a transformation efficiency of 985 CFU/6.8 μg DNA and a gene‐targeting ratio of two among 30 transformants. Copyright © 2017 John Wiley & Sons, Ltd.     

Construction of an autonomously replicating plasmid in n-alkane-assimilating yeast, Candida tropicalis

A transformation system using the autonomously replicating plasmid in the n-alkane-assimilating and asporogenic diploid yeast, Candida tropicalis, was developed. For the cloning of a DNA fragment containing a potential autonomously replicating sequence (ARS) from the genomic DNA of C. tropicalis, the ura3 mutant obtained using ethylmethane sulfonate as the host and the URA3 gene amplified by PCR using the C. tropicalis genomic DNA as a selectable marker were prepared. Comparison of ARSs among yeasts revealed that the consensus sequence found in S. cerevisiae was also present in C. tropicalis. The autonomously replicating plasmid containing the putative ARS as the shuttle vector, capable of replicating in both E. coli and C. tropicalis, was first constructed. The transformation system using this plasmid, in addition to the integrative transformation system, will be applicable to genetic studies of C. tropicalis.     

Cloning and characterization of the peroxisomal acyl CoA oxidase ACO3 gene from the alkane-utilizing yeast Yarrowia lipolytica

The ACO3 gene, which encodes one of the acyl-CoA oxidase isoenzymes, was isolated from the alkane-utilizing yeast Yarrowia lipolytica as a 10 kb genomic fragment. It was sequenced and found to encode a 701-amino acid protein very similar to other ACOs, 67·5% identical to Y. lipolytica Aco1p and about 40% identical to S. cerevisiae Pox1p. Haploid strains with a disrupted allele were able to grow on fatty acids. The levels of acyl-CoA oxidase activity in the ACO3 deleted strain, in an ACO1 deleted strain and in the wild-type strain, suggested that ACO3 encodes a short chain acyl-CoA oxidase isoenzyme. This narrow substrate spectrum was confirmed by expression of Aco3p in E. coli. © 1998 John Wiley & Sons, Ltd.     

A deeper understanding of the spontaneous derepression of the URA3 gene in MaV203 Saccharomyces cerevisiae and its implications for protein engineering and the reverse two-hybrid system

Within the field of protein-based biomaterials, the need exists for both covalent and oriented bioconjugation strategies for improved performance. Such bioconjugation reactions can be facilitated by engineering proteins with chemically activated amino acids at strategically chosen sites. The incorporation of these unnatural amino acids (uAAs) can be achieved by using the nonsense suppression technique. This requires an aminoacyl-tRNA-synthetase (aaRS) that exclusively recognizes the uAA and loads it to the corresponding tRNA. Appropriate (aaRS) mutants can be found through reverse engineering using the Saccharomyces cerevisiae strain MaV203. This strain contains a counterselectable, Gal4p-inducible SPAL10::URA3 fusion and deletions in the endogenous GAL80 and GAL4 genes. Therefore, it has been used extensively for the screening of aaRS mutant libraries. It is generally assumed that the SPAL10 promoter actively represses the URA3 gene in the absence of Gal4p, resulting in MaV203 cells with a Ura ⁻ phenotype. The current contribution reveals that in a small fraction of MaV203 cells, a basal expression of the URA3 gene occurs. The unexpected URA3 expression is reported for the first time, and the nature of the mutation causing this expression was identified as a spontaneous recessive mutation in a single gene of a protein involved in the repression of the SPAL10 promoter. The basal URA3 expression causes aaRS mutants to be missed, which affects the outcome of the library screening. It is demonstrated that the use of diploid cells can circumvent the MaV203 Ura⁺ phenotype, allowing for an optimization of S. cerevisiae library screening.     

Isolation and characterization of Candida kefyr orotidine‐5′‐phosphate decarboxylase (URA3) gene

Candida kefyr is a common yeast species that can be found in fermented milk and cheeses. As a first step to developing a gene transfer system for C. kefyr, the orotidine‐5′‐phosphate decarboxylase (URA3) gene was cloned, using degenerate PCR and genome walking. The uninterrupted open reading frame of the C. kefyr URA3 gene spans 801 bp, corresponding to 267 amino acid residues. The functionality of the gene was confirmed by complementation of ura3 auxotrophs of C. albicans and Saccharomyces cerevisiae. Phylogenetic analysis of the deduced amino acid sequence indicated that it shares a high degree of homology with other Candida URA3 homologues. The GenBank Accession No. of the C. kefyr URA3 gene is FJ914763. Copyright © 2009 John Wiley & Sons, Ltd.     

I. Yeast sequencing reports. Isolation and characterization of the LEU2 gene of Hansenula polymorpha

A DNA fragment carrying the LEU2 gene of methylotrophic yeast Hansenula polymorpha was isolated by complementation of the leuB mutation of Escherichia coli. The nucleotide sequence of the isolated DNA fragment contains an open reading frame of 363 codons, coding for a protein 80% identical to the LEU2 gene product of Saccharomyces cerevisiae. Further downstream, there is a partial reading frame with no obvious similarity to known proteins. The LEU2 gene of H. polymorpha cannot complement the leu2 mutation of S. cerevisiae. The sequence has been entered in the EMBL data library under the Accession Number U00889.     

Non‐homologous end joining‐mediated functional marker selection for DNA cloning in the yeast Kluyveromyces marxianus

The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination‐based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non‐homologous end‐joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C‐terminal‐truncated non‐functional ura3 selection marker and the truncated region were PCR‐amplified separately, mixed and directly used for the transformation. URA3⁺ transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C‐terminal encoding sequence that could restore the function of a truncated non‐functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR‐amplified target DNAs and C‐terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ‐based cloning and recombinant DNA construction. The one‐step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date. Copyright © 2013 John Wiley & Sons, Ltd.     

Isolation and Characterization of the Candida albicans PFY1 Gene for Profilin

We have isolated the Candida albicans gene for profilin, PFY1. Degenerate oligonucleotide primers based on regions of high homology were utilized to obtain a polymerase chain reaction-amplified copy of the gene. This was then used as a probe to isolate the gene from a C. albicans genomic library. Our studies indicate that the full-length gene is unstable in Escherichia coli. Several clones were sequenced, and the predicted amino acid sequence demonstrated homology with profilin proteins from other organisms, most notably Saccharomyces cerevisiae. Northern analysis revealed that the gene is expressed in C. albicans. Attempts to express the gene in S. cerevisiae cells were unsuccessful until the C. albicans promoter was replaced with an S. cerevisiae promoter. Functional complementation of the gene was demonstrated in S. cerevisiae profilin-requiring cells. Antibodies raised to isolated C. albicans profilin protein recognized a protein of the predicted molecular weight when the gene was expressed in S. cerevisiae cells. The sequence of the C. albicans PFY1 gene has been deposited in the Genome Sequence database under Accession Number L3783. © 1997 John Wiley & Sons, Ltd.     

The KlPMR1 gene of Kluyveromyces lactis encodes for a P‐type Ca2+‐ATPase

A novel P‐type Ca²⁺‐ATPase gene has been cloned and sequenced in the yeast Kluyveromyces lactis. The gene has been named KlPMR1 and is localized on chromosome I. The putative gene product contains 936 residues and has a calculated molecular weight of 102 437 Da. Analysis of the deduced amino acid sequence (KlPmr1p) indicated that the encoded protein retains all the highly conserved domains characterizing the P‐type ATPases. KlPmr1p shares 71% amino acid identity with Pmr1p of S. cerevisiae, 62% with HpPmr1p of Hansenula polymorpha, 56% with YlPmr1p of Yarrowia lipolytica and 52% with the Ca²⁺‐ATPase encoded for by the SPCA1 gene of Rattus norvegicus; these similarities place KlPmr1p in the SPCA group (secretory pathway Ca²⁺‐ATPase) of the P‐type ATPases. The K. lactis strain harbouring the Klpmr1 disrupted gene is not able to grow in presence of low calcium concentrations and shows hypersensitivity to high concentrations of EGTA in the medium. These defects are relieved by PMR1 of S. cerevisiae on a centromeric plasmid, demonstrating that KlPMR1 encodes for a functional Pmr1p homologue. The sequence described can be retrieved under EMBL Accession No. AJ001018. Copyright © 1999 John Wiley & Sons, Ltd.