Heterologous gene expression of the glyphosate resistance marker and its application in yeast transformation

Summary The E. coli aroA gene was inserted between yeast promoter and terminator sequences in different shuttle expression plasmids and found to confer enhanced EPSP synthase activity as well as resistance to glyphosate toxicity. Subsequently, a transformation system using these newly constructed vectors in yeast was characterized. The efficiency of the glyphosate resistance marker for transformation and selection with plasmid pHR6/20-1 in S. cerevisiae laboratory strain SHY2 was found to be relatively high when compared with selection for LEU2 prototrophy. The fate of the recombinant plasmid pHR6/20-1 in the transformants, the preservation of the aroA E. coli DNA fragment in yeast, mitotic stability, EPSP synthase activity, and growth on glyphosate-containing medium have been investigated. As this plasmid also allows direct selection for glyphosate resistant transformants on rich media, the glyphosate resistance marker was used for transforming both S. cerevisiae laboratory strain SHY2 and brewer's yeast strains S. cerevisiae var. uvarum BHS5 and BHS2. In all cases, the vector pHR6/20-1 was maintained as an autonomously replicating plasmid. The resistance marker is, therefore, suitable for transforming genetically unlabeled S. cerevisiae laboratory, wild, and industrial yeast strains.Abbreviations EPSP 5-enolpyruvylshikimate 3-phosphate     

A color-selectable and site-specific integrative transformation system for gene expression studies in the dematiaceous fungus Wangiella (Exophiala) dermatitidis

To explore potential virulence factors in the dematiaceous (melanized) fungus Wangiella dermatitidis, we established a gene expression system with properties of homologous transformation and color identification. Using a polyketide synthase gene (WdPKS1) fragment for targeting, we found that 52% of transformants became albinos easily distinguishable from nonspecific transformants. Southern analysis confirmed that the integrations were at the WdPKS1 locus, which however did not affect transformant growth. With a heterologous promoter, P-glaA, enhanced expression of lacZ was found at 37 °C. Our results indicated that this system allows the efficient production of isogenic strains for gene function analysis in W. dermatitidis. Received: 12 March / 18 June 1999     

Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha

Summary For the transformation of the yeast Hansenula polymorpha we have constructed a set of hybrid plasmids carrying the LEU2 gene of Saccharomyces cerevisiae as a selective marker and fragments of mitochondrial DNA of Candida utilis and H. polymorpha or chromosomal DNA fragments of H. polymorpha as replicator sequences. The replication properties of chimeric plasmids in the yeast H. polymorpha were investigated. We showed that for plasmids propagated autonomously in this yeast the plasmid monomers could be detected in the transformants only during the immediate time after the transformation event. Further growth under selective conditions led to the selection of polymeric forms of plasmid DNA as it was clearly shown for transformants carrying cosmid pL2 with mtDNA fragment of C. utilis. Such transformants carrying polymerized plasmids showed a remarkably increased stability of the transformed phenotype. Cosmid pL2 was able to shuttle between Escherichia coli, S. cerevisiae and H. polymorpha, whereas plasmids with DNA fragments from H. polymorpha did not transform S. cerevisiae effectively.     

Bialaphos resistance as a dominant selectable marker in Neurospora crassa

Summary Conidia of Neurospora crassa are sensitive to the herbicide bialaphos at concentrations of 160 mg/1 in Westergaard's or Fries' minimal media. Plasmid pJA4 was constructed by inserting a truncated bar gene from Streptomyces hygroscopicus fused to the his-3 promoter from N. crassa into pUC19. The bar gene in plasmid pJA4 confers resistance to bialaphos when transformants are selected on a medium containing bialaphos. The bar gene can be used as an additional dominant selectable marker for transformation of fungi.     

The <Emphasis Type="Italic">Aspergillus nidulans amdS</Emphasis> gene as a marker for the identification of multicopy T-DNA integration events in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Aspergillus awamori</Emphasis>

The Aspergillus nidulans amdS selection marker was used for the identification of multicopy T-DNA insertions in Agrobacterium-mediated transformation of Asp. awamori. The selection of transformants on agar plates containing acetamide as sole nitrogen source and hygromycin resulted in a six-fold decrease in the transformation frequency, compared with the transformation frequency obtained after hygromycin selection alone. However, it was found that 47% of the transformants obtained after hygromycin and acetamide double selection contained multiple T-DNA integrations. Furthermore, it was found that the multicopy transformants could easily be identified based on their growth rate on agar plates containing acetamide medium. Based on these data, it can be concluded that the amdS marker can also be used as a selection marker in Agrobacterium-mediated transformation of Asp. awamori and that it is a very useful marker to identify those transformants containing multiple T-DNA integrations.     

Integrative transformation of the yeast Yarrowia lipolytica

Summary An EcoR1 shotgun of Yarrowia lipolytica DNA was inserted into the plasmid YIp333 which carries the LYS2 gene of S. cerevisiae. The resulting plasmid pool was transformed in both S. cerevisiae and Y. lipolytica. Whereas numerous replicating plasmids could be isolated from the S. cerevisiae Lys⁺ transformants, all transformants of Y. lipolytica so far analyzed were found to result from integrative transformation. This occurred at a frequency of 1 to 10 transformants per g of input DNA. Co-transformation occurred at high frequency and resulted in tandem integration of 2 to 10 copies of the incoming DNA. Structural and segregational stability of the transforming DNA were both high.     

Carboxin resistance transformation of the homobasidiomycete fungus Pleurotus ostreatus

A novel selection marker gene for transformation of the white-rot basidiomycete Pleurotus ostreatus was developed by introducing a point mutation in a gene which encodes the iron-sulfur protein (Ip) subunit of succinate dehydrogenase. The mutant gene, Cbx ^R, encodes a modified Ip subunit with an amino-acid substitution (His239 to Leu) and confers resistance to the systemic fungicide, carboxin. The DNA sequence was integrated ectopically in the chromosome of the transformants. This is the first report of a homologous marker gene which is available for the molecular breeding of an edible mushroom. Received: 24 August / 1 December 1999     

Insertional mutagenesis in Coprinus cinereus: use of a dominant selectable marker to generate tagged, sporulation-defective mutants

We have constructed a dominant selectable marker, PHT1, for transformation of the basidiomycete Coprinus cinereus. PHT1 consists of a bacterial hygromycin B resistance gene fused to the promoter and terminator regions of the C. cinereusβ-tubulin gene. We found in transformation experiments that PHT1 confers hygromycin B resistance to all strains of C. cinereus tested, that it integrates without apparent bias into the genome, and that it is stable through meiotic crosses. We used a plasmid containing this marker, pPHT1, for restriction enzyme-mediated integration (REMI) and found that this technique could increase transformation efficiencies more than seven-fold. In REMI experiments using KpnI, the integrated DNA was flanked by intact KpnI sites in 53% of the cases examined, single-copy insertions represented 60% of the integration events, and most multicopy insertions were oriented head-to-tail. A screen of REMI-generated transformants yielded sporulation-defective mutants at a frequency of 1.2%. Genetic analysis showed that in six of nine mutants examined, the defect in spore formation is most likely a direct result of the pPHT1 insertion, and in three of these mutants a single pPHT1 locus was shown to cosegregate with the sporulation defect. We used semi-random PCR to isolate the genomic DNA adjacent to one pPHT1 insertion in a sporulation-defective mutant and found that we had disrupted the C. cinereusspo11 gene. Thus, REMI, in combination with pPHT1, is a powerful tool for the dissection of the meiotic process in C. cinereus. Received: 26 February / 13 July 1999     

Genetic transformation of Ascochyta rabiei using Agrobacterium-mediated transformation

In order to study pathogenic mechanisms of the plant pathogen Ascochyta rabiei, conditions for efficient transformation using Agrobacterium-mediated transformation were investigated. Hygromycin B resistance (hph) was superior to geneticin resistance (nptII) for selecting transformants, and the hph gene was more efficiently expressed by the Aspergillus nidulans trpC promoter than by the Cauliflower mosaic virus 35S promoter CaMV35S. Co-cultivation on solid media for 72 h was optimal for generating transformants, but increasing the ratio of bacterial cells to conidia did not affect transformation efficiency. All hygromycin B-resistant transformants carried transfer-DNA (T-DNA) as determined by polymerase chain reaction (PCR) and the T-DNA integrations appeared to be random and in single copy as detected by Southern hybridization. Transformants remained resistant to hygromycin B in the absence of selection. Variations in colony morphology were observed in the presence of hygromycin B under different culture conditions, and a variety of altered phenotypes including reduced virulence were observed among 550 transformants. Inverse PCR was more efficient than TAIL-PCR in identifying flanking genomic sequences from T-DNA borders, and the possible causes are discussed. This transformation technique and recovery of flanking DNA using inverse PCR will provide a useful tool for genetic studies of A. rabiei.     

Non-selective transformation of Saccharomyces cerevisiae

Summary Wild or industrial yeast strains cannot be transformed by most selective vectors due to a lack of auxotrophic mutations. To enable identification of transformants of such yeast species, we have developed a 2-µm DNA vector with an indicator gene that can be used without any additional marker. The Escherichia coli gene for -lactamase (bla) was placed under the control of the yeast promoter for the structural gene encoding ADHI. This increased the amount of -lactamase produced in Saccharomyces cerevisiae 100-fold giving an enzyme activity in transformant colonies which is high enough to be detected directly on indicator plates. Non-selectively, the transformation frequency is even higher than under selective conditions indicating that selection does not assist the establishment of new plasmids. Transformants isolated non-selectively were found to retain the endogenous 2-µm DNA. Under control of appropriate promoters, the bacterial bla gene may also provide a convenient marker for other eukaryotic transformation systems.     

High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier

Summary A method, using LiAc to yield competent cells, is described that increased the efficiency of genetic transformation of intact cells of Saccharomyces cerevisiae to more than 1 × 10⁵ transformants per microgram of vector DNA and to 1.5% transformants per viable cell. The use of single stranded, or heat denaturated double stranded, nucleic acids as carrier resulted in about a 100 fold higher frequency of transformation with plasmids containing the 2m origin of replication. Single stranded DNA seems to be responsible for the effect since M13 single stranded DNA, as well as RNA, was effective. Boiled carrier DNA did not yield any increased transformation efficiency using spheroplast formation to induce DNA uptake, indicating a difference in the mechanism of transformation with the two methods.     

Transformation of the bioherbicide Colletotrichum gloeosporioides f. sp. aeschynomene by electroporation of germinated conidia

A highly efficient and reproducible procedure for the transformation of the bioherbicide Colletotrichum gloeosporioides f. sp. aeschynomene by electroporation of germinated conidia is reported. Development of the procedure involved a detailed study of the germination process followed by extensive calibration of the electrical-pulse and selection conditions. Optimization of the transformation protocol was facilitated by the use of the green fluorescent protein that helped in the identification of stable transformants and in a fast assessment of transgene expression levels, colony homogeneity and genetic stability. Under optimal conditions, over 80 stable transformants/cuvette were obtained. Plasmid integration was predominantly homologous, but high transformation rates were obtained both with homologous and non-homologous vectors. The method described not only opens up opportunities for the genetic manipulation of C. gloeosporioides f. sp. aeschynomene, but also provides a framework for the development and optimization of transformation in other fungi. Received: 27 November 1998 / 6 March 1999     

<Emphasis Type="Italic">KNQ1</Emphasis>, a<Emphasis Type="Italic"> Kluyveromyces lactis</Emphasis> gene encoding a drug efflux permease

Several transport systems play an important role in conferring multiple drug resistance, presumably due to their catalysis of the energy-dependent extrusion of a large number of structurally and functionally unrelated compounds out of the cells. In the present work, the gene named KNQ1 (encoding Kluyveromyces lactis membrane permease) was cloned by functional complementation of the cycloheximide-hypersensitivity phenotype of the Saccharomyces cerevisiae mutant strain lacking a functional PDR5 gene. The isolated gene exhibited 48.9% identity with the S. cerevisiae ATR1 gene conferring resistance to aminotriazole and 4-nitroquinoline-N-oxide and encoded a protein of 553 amino acids. When present in multicopy, it efficiently complemented the phenotype associated with the pdr5 or pdr1pdr3 mutations in S. cerevisiae. Overexpression of the KNQ1 gene in K. lactis wild-type strains led to resistance against several cytotoxic compounds, like 4-nitroquinoline-N-oxide, 3-aminotriazole, bifonazole and ketoconazole. The gene was assigned to K. lactis chromosome III and its expression was found to be responsive to oxidative stress induced by hydrogen peroxide. Based on the phenotype of homologous and heterologous transformants, we propose that the gene encodes a membrane-associated component of the machinery responsible for decreasing the concentration of several toxic compounds in the cytoplasm of yeast cells.Communicated by K. Breunig     

Shuttle vectors for <Emphasis Type="Italic">Candida albicans</Emphasis>: control of plasmid copy number and elevated expression of cloned genes

Plasmids containing the inosine monophosphate dehydrogenase gene CaIMH3 from Candida albicans strain ATCC 32354 transform their host to resistance against mycophenolic acid (MPA). The transformants maintain the plasmids at a high copy number (20–40 per cell) and express the CaIMH3 gene at very high levels relative to untransformed controls. The plasmid copy number can be controlled by the concentration of MPA in the media. The transformation procedure is reproducible and the efficiency of transformation is high, up to 15,000 per microgram. Unrearranged plasmids are readily recovered by transforming total DNA from transformants back into Escherichia coli. C. albicans genes cloned into the plasmid are expressed at elevated levels relative to untransformed controls. A derivative vector containing the CaMAL2 promoter and termination sequences expresses the CaERG11 ORF at high levels and confers moderate resistance to fluconazole. These shuttle vectors should facilitate global genomics approaches in C. albicans that have been hampered by its diploid genome.     

Transformation of the thermophilic fungus Humicola grisea var. thermoidea and overproduction of Humicola glucoamylase

Summary The thermophilic fungus Humicola grisea var. thermoidea was successfully transformed using a bleomycin-phleomycin resistance gene linked to regulatory sequences from Aspergillus nidulans. Transformation was achieved using the lithium acetate method with young mycelia, and transformants were obtained at a frequency of 0.5–2 per g of plasmid DNA. Vector DNA used in transformations was integrated in the genome of Humicola in varying patterns and copy number, and transformants were mitotically stable. Extra copies of an Humicola gla1 gene encoding glucoamylase (GAM) were introduced into the genome of several Humicola strains by transformation, with the result that some transformants produced almost 3-fold more GAM in comparison to the untransformed parental strains.     

Glutathione depletion leads to delayed growth stasis in Saccharomyces cerevisiae : evidence of a partially overlapping role for thioredoxin

 Disruption of the first enzyme of glutathione biosynthesis in both Saccharomyces cerevisiae and Schizosaccharomyces pombe leads to a glutathione auxotrophy phenotype on plates. However, growth experiments in liquid medium revealed that the cessation of growth resulting from glutathione depletion in these yeasts is very delayed in S. cerevisiae compared to S. pombe. Glutathione metabolism was investigated to understand this delayed growth stasis in S. cerevisiae. The assimilation of reduced and oxidized glutathione, the intracellular storage pools of glutathione and the turnover of this compound were investigated and found to be similar in both yeasts. A possible overlapping role of intracellular thioredoxin in causing delayed stasis was studied. Yeast thioredoxin was overexpressed in S. cerevisiae and was found to partially relieve the dependence of S. cerevisiae glutathione auxotrophs on extracellular glutathione in glucose-grown cultures, as well as in glycerol-grown cultures where conditions of increased glutathione requirements exists in the cell. By partially, but not completely, compensating for glutathione deficiency in this yeast, thioredoxin thus appeared to be the major factor that was causing the delayed growth stasis following glutathione depletion in this yeast. Received: 16 November 1999 / 15 February 2000     

Molecular characterization of a chromosomal rearrangement involved in the adaptive evolution of yeast strains

Wine yeast strains show a high level of chromosome length polymorphism. This polymorphism is mainly generated by illegitimate recombination mediated by Ty transposons or subtelomeric repeated sequences. We have found, however, that the SSU1-R allele, which confers sulfite resistance to yeast cells, is the product of a reciprocal translocation between chromosomes VIII and XVI due to unequal crossing-over mediated by microhomology between very short sequences on the 5' upstream regions of the SSU1 and ECM34 genes. We also show that this translocation is only present in wine yeast strains, suggesting that the use for millennia of sulfite as a preservative in wine production could have favored its selection. This is the first time that a gross chromosomal rearrangement is shown to be involved in the adaptive evolution of Saccharomyces cerevisiae.     

Expression of the Escherichia coli β-glucuronidase gene in industrial and phytopathogenic filamentous fungi

Summary The plant pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed using two positive selection systems, one based on the Escherichia coli hygromycin B phosphotransferase gene (hph) and the other on the Neurospora crassa -tubulin gene bml which encodes resistance to the methyl benzimidazole carbamate fungicides. Both selection systems gave a transformation frequency of 1–20 transformants g^–1 DNA. The vector DNA was integrated into the genome and the number and sites of integration varied among the transformants. The hph transformants were mitotically stable and the transformed gene was transmitted through spores. In contrast the bml transformants were less stable.     

Use of the Tn903 neomycin-resistance gene for promoter analysis in the fission yeast Schizosaccharomyces pombe

Summary The bacterial neo gene from transposon Tn903 (Tn601) was used for dominant transformation of the fission yeast Schizosaccharomyces pombe. It was found that high transformation efficiency was dependent on a high level of promoter activity, mediated by the strong promoter of the Schizosaccharomyces pombe alcohol dehydrogenase gene (adh1), as shown by comparing the efficiency of transformation to G418-resistance, the resistance levels of transformed cells, and the in vitro aminoglycoside phosphotransferase activity. On the other hand, the heterologous promoter of the Saccharomyces cerevisiae alcohol dehydrogenase I gene (adc1) is shown to be a weak promoter in Schizosaccharomyces pombe, though its activity is significantly enhanced in cells grown on glycerol as a carbon source. This system for selection and detection of promoter-active sequences may provide a useful basis for the analysis of promoter elements in fission yeast.