A disruption-replacement approach for the targeted integration of foreign genes in Hansenula polymorpha

A system has been developed which allows the selection of integrative transformants with replacement of the Hansenula polymorpha methanol oxidase gene (MOX) with expression cassettes carrying heterologous gene under the control of the MOX promoter. The system is convenient for comparison of the expression levels of different constructs integrated into the same locus of the H. polymorpha genome. This system was used to compare the secretion levels of human urinary plasminogen activator, the secretion of which was directed by different signal sequences.     

Vectors and gene targeting modules for tandem affinity purification in Schizosaccharomyces pombe

We describe the construction of tagging cassettes and plasmids for tandem affinity purification (TAP) of proteins in Schizosaccharomyces pombe. The tagging cassettes are designed for either carboxy- or amino-terminal tagging of proteins. The carboxyl terminal tags differ in that they contain either two or four repeats of IgG binding units. For tagging endogenous loci, the cassettes contain the kan MX6 module to allow for selection of G418-resistant cells. The amino-terminal tagging vectors allow for the regulated expression of proteins. Sz. pombe Cdc2p was chosen to test these new affinity tags. Several known binding proteins co-purified with both Cdc2p-CTAP and N-TAP-Cdc2p, indicating the usefulness of these tags for the rapid purification of stable protein complexes from Sz. pombe.     

Disruption and basic functional analysis of five chromosome X novel ORFs of Saccharomyces cerevisiae reveals YJL125c as an essential gene for vegetative growth

We describe the disruption and basic functional analysis of five novel open reading frames (ORFs) discovered during the sequencing of the Saccharomyces cerevisiae genome: YJL118w, YJL122w, YJL123c, YJL124c, YJL125c, located on chromosome X. Disruptions have been realized using the long-flanking homology-PCR replacement strategy (LFH-PCR; Wach et al., 1996) in the FY1679 diploid strain. Sporulation and tetrad analysis of these heterozygous deletants were performed, as well as a functional analysis on the haploid deleted strains: different growth conditions (complete glucose and glycerol, minimal media) at three temperatures 15, 30 and 37 degrees C were tested. Analysis revealed YJL125c as an essential gene; the four other ORFs were non-essential and showed no particular phenotype. In addition, the five kanMX4 disruption cassettes were cloned in pUG7 vector. Finally, the five ORFs with their promoter and terminator regions were cloned in the centromeric yeast vector pRS416. The vectors containing the disruption cassettes, the cognate wild-type genes, as well as the deletant strains are available at the EU EUROFAN (EUROSCARF, Frankfurt, DE) genetic and stock centre.     

A truncated derivative of nmt 1 promoter exhibits temperature-dependent induction of gene expression in Schizosaccharomyces pombe

Despite increasing exploitation of Schizosaccharomyces pombe as a model system there is a lack of convenient vectors for research and application. Expression with the commonly used promoter, nmt 1, requires a laborious regime involving the removal of repressor, thiamine, from a growing culture and further growth for 18 h to achieve maximum expression, thus underlining the need for more user-friendly promoters. We report here the isolation and characterization of a truncated derivative of the nmt 1 promoter having novel induction characteristics: it is induced by shift of growth temperature from 36 degrees C to 25 degrees C, achieving maximum expression within 3 h. Similar features of expression were observed with the reporter genes GFP and beta-galactosidase, a native gene, cdc 18, and a commercially important foreign therapeutic protein, streptokinase. The new promoter element offers additional advantages, such as lack of deleterious effect on cell viability and potential ability to express toxic proteins. These features make the new promoter a potentially better alternative to nmt 1, both as a research tool and for expression of commercially important proteins in Sz. pombe, and suggest the possibility of using similar approaches to design promoters with novel and useful properties.     

New generation of loxP‐mutated deletion cassettes for the genetic manipulation of yeast natural isolates

In this work, we developed molecular tools used in standard laboratory yeast strains, such as the cre–loxP system, so that they can be used with natural and industrial prototrophic yeast species. We constructed a new generation of dominant cassettes, with mutated loxP sites (loxLE and lox2272) and selectable drug markers, to create heterothallic strains and auxotrophic mutants without incurring in the risk of generating chromosomal rearrangements. We have shown that our newly developed loxLE–hphNT1–loxRE and lox2272–natNT2–lox2272 gene‐disruption cassettes can be present in the yeast genome together with the widely used loxP–marker gene–loxP cassettes without any recombination between the lox sequences. Moreover, we also developed a new phleomycin‐resistant Cre‐expressing vector (to excise multiple markers simultaneously) and two new standard loxP deletion cassettes containing hygromicin B and cloNAT as selecatable markers. To validate these cassettes, we created heterothallic auxotrophic S. cerevisiae strains, without the risk of incurring gross chromosomal rearrangements, and we showed an example of a fitness study of intraspecific hybrids deriving from parents with different adaptations to carbon‐limited resources. Copyright © 2010 John Wiley & Sons, Ltd.     

Development of a semi-quantitative plate-based alpha-galactosidase gene reporter for Schizosaccharomyces pombe and its use to isolate a constitutively active Mam2

To extend the tools available for biochemical and genetical analysis in the fission yeast Schizosaccharomyces pombe we have investigated the development of gene reporter systems using the secreted alpha-galactosidase encoded by the Sz. pombe ORF SPAC869.07c (CAB60017), which we propose naming Mel1p to reflect its structural and functional similarity to MEL1p in Saccharomyces cerevisiae. The alpha-galactosidase activity can be monitored in liquid assays and converted the colourless substrate 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside (X-alpha-gal) into an insoluble blue product that was suitable for semi quantitative plate-based assays; colonies expressing the highest levels of alpha-galactosidase developed the most intense blue colour. Unlike assays based on beta-galactosidase, the Sz. pombe colonies develop the blue colouration under normal growth conditions, avoiding the need to replicate colonies to fresh plates for analysis. It is therefore suitable for screening large numbers of colonies. To illustrate the use of mel1 as a reporter we linked expression to the sxa2 gene promoter to provide a convenient readout for signalling through the pheromone response pathway. The sxa2 > mel1 strain identified constitutively active Mam2 pheromone receptors from a randomly mutagenised library. There was an approximate correlation between the intensity of the blue colour developed by each mutant colony and its level of constitutive activity and we identified a subset of mutants with low constitutive activity that could not have been isolated by a previous screen using nutritional selection. The mel1 alpha-galactosidase activity identified and characterised in this study can be easily adapted to provide a gene reporter for many biological processes and is a new addition to the research tools available in Sz. pombe.     

Three novel antibiotic marker cassettes for gene disruption and marker switching in Schizosaccharomyces pombe

The ease of construction of multiple mutant strains in Schizosaccharomyces pombe is limited by the number of available genetic markers. We describe here three new cassettes for PCR-mediated gene disruption that can be used in combination with commonly used fission yeast markers to make multiple gene deletions. The natMX6, hphMX6 and bleMX6 markers give rise to resistance towards the antibiotics nourseothricin (NAT), hygromycin B and phleomycin, respectively. The cassettes are composed of exogenous sequences to increase the frequency of integration at targeted loci, and have a structure similar to the commonly used pFA6a-kanMX6 modular plasmid system. This allows a simple exchange of the kanMX6 marker in existing strains with any of the three new cassettes. Alternatively, oligonucleotide primers designed for the modular kanMX6 cassettes can be used to make the transforming PCR fragments for gene disruption. We illustrate the construction of a mutant strain with six independent gene disruptions, using the novel antibiotic cassettes in combination with existing genetic markers.     

A two-step cloning-free PCR-based method for the deletion of genes in the opportunistic pathogenic yeast Candida lusitaniae

We describe a new cloning-free strategy to delete genes in the opportunistic pathogenic yeast Candida lusitaniae. We first constructed two ura3 Δ strains in C. lusitaniae for their use in transformation experiments. One was deleted for the entire URA3 coding sequence; the other possessed a partial deletion within the coding region, which was used to determine the minimum amount of homology required for efficient homologous recombination by double crossing-over of a linear DNA fragment restoring URA3 expression. This amount was estimated to 200 bp on each side of the DNA fragment. These data constituted the basis of the development of a strategy to construct DNA cassettes for gene deletion by a cloning-free overlapping PCR method. Two cassettes were necessary in two successive transformation steps for the complete removal of a gene of interest. As an example, we report here the deletion of the LEU2 gene. The first cassette was constituted by the URA3 gene flanked by two large fragments (500 bp) homologous to the 5' and 3' non-coding regions of LEU2. After transformation of an ura3 Δ recipient strain and integration of the cassette at the LEU2 locus, the URA3 gene was removed by a second transformation round with a DNA cassette made by the fusion between the 5' and 3' non-coding regions of the LEU2 gene. The overall procedure takes less than 2 weeks and allows the creation of a clean null mutant that retains no foreign DNA sequence integrated in its genome.     

Positive and negative selection LYS5MX gene replacement cassettes for use in Saccharomyces cerevisiae

Novel MX cassettes are described that contain the open reading frames (ORFs) of Saccharomyces cerevisiae or Candida albicans LYS5. The LYS5MX and CaLYS5MX cassettes, the targeting efficiencies of which are equivalent to those of other MX cassettes, are positively selected for Lys+ in a lys5 background. Unlike most of the other MX cassettes, the LYS5MX cassettes are also negatively selectable (alpha-aminoadipate-resistant), which will allow the use of the LYS5MX cassettes in plasmid shuffling and will also greatly facilitate marker recycling.     

A versatile set of vectors for constitutive and regulated gene expression in Pichia pastoris

The budding yeast Pichia pastoris is an attractive system for exploring certain questions in cell biology, but experimental use of this organism has been limited by a lack of convenient expression vectors. Here we describe a set of compact vectors that should allow for the expression of a wide range of endogenous or foreign genes in P. pastoris. A gene of interest is inserted into a modified pUC19 polylinker; targeted integration into the genome then results in stable and uniform expression of this gene. The utility of these vectors was illustrated by expressing the bacterial β-glucuronidase (GUS) gene. Constitutive GUS expression was obtained with the strong GAP promoter or the moderate YPT1 promoter. The regulatable AOX1 promoter yielded very strong GUS expression in methanol-grown cells, negligible expression in glucose-grown cells, and intermediate expression in mannitol-grown cells. GenBank Accession Numbers are: pIB1, AF027958; pIB2, AF027959; pIB3, AF027960; pIB4, AF027961. © 1998 John Wiley & Sons, Ltd.     

丝状真菌基因敲除技术研究进展

丝状真菌在自然界分布广泛，与人类的生产、生活密切相关。近年来，对于其基因功能的研究取得了较大的进展，一系列转化和基因操作技术已在不同的丝状真菌中得到运用。许多对于工业、农业和医药卫生具有重要意义的丝状真菌已经完成或正在进行全基因组序列测定。作者简述了丝状真菌基因敲除的历史，重点介绍了近年来基因敲除技术在丝状真菌研究中的进展，并对冀在丝状真菌基因功能研究、工业菌株改良等方面进行了展望。     

The alcohol dehydrogenase system in the yeast, Kluyveromyces lactis

We have studied the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis. Southern hybridization to the Saccharomyces cerevisiae ADH2 gene indicates four probable structural ADH genes in K. lactis. Two of these genes have been isolated from a genomic bank by hybridization to ADH2. The nucleotide sequence of one of these genes shows 80% and 50% sequence identity to the ADH genes of S. cerevisiae and Schizosaccharomyces pombe respectively. One K. lactis ADH gene is preferentially expressed in glucose-grown cells and, in analogy to S. cerevisiae, was named K1ADH1. The other gene, homologous to K1ADH1 in sequence, shows an amino-terminal extension which displays all of the characteristics of a mitochondrial targeting presequence. We named this gene K1ADH3. The two genes have been localized on different chromosomes by Southern hybridization to an orthogonal-field-alternation gel electrophoresis-resolved K. lactis genome. ADH activities resolved by gel electrophoresis revealed several ADH isozymes which are differently expressed in K. lactis cells depending on the carbon source.     

Cytosine deaminase MX cassettes as positive/negative selectable markers in Saccharomyces cerevisiae

We describe positive/negative selectable cytosine deaminase MX cassettes for use in Saccharomyces cerevisiae. The basis of positive selection for cytosine deaminase (Fcy1) activity is that (a) fcy1 strains are unable to grow on medium containing cytosine as a sole nitrogen source and (b) fcy1 ura3 strains are unable to grow on medium containing cytosine as the sole pyrimidine source. Conversely, as 5-fluorocytosine (5FC) is toxic to cytosine deaminase-producing cells, fcy1 strains are resistant to 5FC. FCY1MX and FCA1MX cassettes, containing open reading frames (ORFs) of S. cerevisiae FCY1 and Candida albicans FCA1, respectively, were constructed and used to disrupt targeted genes in S. cerevisiae fcy1 strains. In addition, new direct repeat cassettes, kanPR, FCA1PR, FCY1PR and CaURA3PR, were developed to allow efficient deletion of target genes in cells containing MX3 repeats. Finally, the FCY1- and FCA1MX3 or PR direct repeat cassettes can be readily recycled after 5FC counter-selection on both synthetic and rich media.     

A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes

Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae. This strategy directs the amplified tags to the desired chromosomal loci due to flanking homologous sequences provided by the PCR-primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C-terminal tagged genes or for the exchange of the promoter and N-terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C-terminal epitope tags as well as nine different promoter substitutions in combination with N-terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast-optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N- and C-terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost-effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches.     

Construction and evaluation of gRNA arrays for multiplex CRISPR-Cas9

Endonuclease system CRISPR-Cas9 represents a powerful toolbox for the budding yeast's Saccharomyces cerevisiae genome perturbation. The resulting double-strand breaks are preferentially repaired via highly efficient homologous recombination, which subsequently leads to marker-free genome editing. The goal of this study was to evaluate precise targeting of multiple loci simultaneously. To construct an array of independently expressing guide RNAs (gRNAs), the genes encoding them were assembled through a BioBrick construction procedure. We designed a multiplex CRISPR-Cas9 system for targeting 6 marker genes, whereby the gRNA array was expressed from a single plasmid. To evaluate the performance of the gRNA array, the activity of the designed system was assessed by the success rate of the introduction of perturbations within the target loci: successful gRNA expression, followed by target DNA double-strand breaks formation and their repair by homologous recombination led to premature termination of the coding sequence of the marker genes, resulting in the prevention of growth of the transformants on the corresponding selection media. In conclusion, we successfully introduced up to five simultaneous perturbations within single cells of yeast S. cerevisiae using the multiplex CRISPR-Cas9 system. While this has been done before, we here present an alternative sequential BioBrick assembly with the capability to accommodate many highly similar gRNA-expression cassettes, and an exhaustive evaluation of their performance.     

Accumulative gene integration into a pre-determined site using Cre/loxP

Site-specific gene recombination systems, such as Cre/loxP, have been used for genetic modification of cells and organisms in both basic and applied research. We previously developed an accumulative gene integration system (AGIS), in which target gene cassettes could be repeatedly integrated into a pre-determined site on a plasmid or cellular genome by recombinase-mediated cassette exchange (RMCE), using Cre and mutated loxPs. In the present study, we designed a simplified AGIS. For gene integration into a target site, the previous system used two loxP sites in the acceptor DNA, whereas the new system uses a single loxP site. The gene integration reactions were repeated four times in vitro using Cre protein and specific plasmids. The expected integration reactions mediated by Cre occurred at the loxP sites, resulting in integration of four target genes. The system was also used for genomic integration of reporter genes using Chinese hamster ovary (CHO) cells. The reporter genes were efficiently introduced into the CHO genome in a Cre-dependent manner, and transgene expression was detected after the integration reaction. The expression levels of the reporter genes were enhanced, corresponding to the increase of transgene copy number. Recombinase-mediated AGIS provides a useful tool for the modification of cellular genomes.     

A novel kanamycin/G418 resistance marker for direct selection of transformants in Escherichia coli and different yeast species

We have developed a set of cloning vectors possessing a modified Tn903 kanamycin resistance gene that enables the selection of both kanamycin‐resistant transformants in Escherichia coli and G418‐resistant transformants in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha and Pichia pastoris. Expression of this gene in yeast is controlled by the H. polymorpha glyceraldehyde‐3‐phosphate dehydrogenase promoter, while expression in E. coli is governed by an upstream E. coli lacZ promoter. Applicability of the vectors for gene disruption in H. polymorpha and S. cerevisiae was demonstrated by inactivation of the HpMAL1 and URA3 genes, respectively. One of the vectors possesses a H. polymorpha ARS allowing plasmid maintenance in an episomal state. The small size of the vectors (2–2.5 kb) makes them convenient for routine DNA cloning. In addition, we report a novel approach for construction of gene disruption cassettes. Copyright © 2009 John Wiley & Sons, Ltd.     

Disruption of six ORFs on Saccharomyces cerevisiae chromosome X: the YJL069c gene of unknown function is essential to cell viability.

The short flanking homology PCR strategy (Wach et al., 1994) was used to disrupt six open reading frames (ORFs) on chromosome X of diploid strains (FY1679 and W303) of the yeast Saccharomyces cerevisiae. Two of the six ORFs analysed (YJL069c and YJL066c) display no similarity to known sequences. Three others (YJL065c, YJL068c, and YJL070c) are similar to those respectively encoding the DNA polymerase epsilon subunit c, human esterase D and rat AMP deaminase 1. YJL071w has recently been identified as the ARG2 gene coding for acetylglutamate synthase. Inactivation of the YJL069c gene proved lethal and the yjl071w haploid disruptants were auxotrophic for arginine. For the four other gene inactivations, neither the heterozygous deletion diploids nor the corresponding haploid deletion mutants displayed any special phenotype when grown on rich glycerol or glucose medium or on synthetic minimal medium at three different temperatures, or on media containing compounds interfering with nucleic acid or protein synthesis. Mating and sporulation efficiencies were the same for the viable disruptants as for wild-type cells. The six kanMX4 disruption cassettes were cloned into the pUG7 vector and each of the cognate wild-type genes was inserted into the pRS416 centromeric plasmid. All strains and plasmids have been deposited in the EUROFAN collection (EUROSCARF, K. -D. Entian, Frankfurt, Germany).