Invertase gene (SUC2) of Saccharomyces cerevisiae as a dominant marker for transformation of Pichia pastoris

A two-step method for the selection of transformants of prototrophic industrial strains of the methylotrophic yeast Pichia pastoris has been developed. This method is based on our observation that P. pastoris cannot use sucrose as the sole carbon source (Suc-) and that introduction of the invertase gene (SUC2) of Saccharomyces cerevisiae renders P. pastoris Suc+. P. pastoris was transformed with a plasmid which contains the SUC2 gene of S. cerevisiae and an autonomously replicating sequence PARS1 from P. pastoris. The transformants were initially allowed to regenerate on medium containing dextrose and the regenerated cells were pooled and plated on sucrose medium to screen for Suc+ transformants. It was shown that the Suc+ transformants of P. pastoris with the autonomously replicating plasmid were highly unstable with respect to the plasmid maintenance, even when grown on sucrose as the sole carbon and energy source. This high instability was attributed to an efficient cross-feeding by Suc- segregants on glucose and fructose generated due to hydrolysis of sucrose by the invertase enzyme secreted by Suc+ cells. Spontaneous integration of the plasmid DNA resulting in a stable Suc+ phenotype was also observed. However, stable Suc+ transformants were obtained more readily by integration of SUC2 into P. pastoris genome following transformation with a linearized plasmid with the ends homologous to P. pastoris HIS4 locus. All such integrants were completely stable for Suc+ phenotype after 20 generations of growth in a nonselective medium.     

A dominant nuclear streptomycin resistance marker for plant cell transformation

Summary Plant cells in photoheterotrophic culture respond to streptomycin by bleaching and retarded growth but no cell death. A new genetic marker for plant cell transformation has been developed that is based on the expression of the enzyme streptomycin phosphotransferase (SPT), and confers the ability to form green colonies on a selective medium. Coding sequences of SPT from the bacterial transposon Tn5 were placed under the control of gene expression signals derived from the Agrobacterium Ti plasmid Ach5. The 5 end of the SPT gene has been replaced with the promoter region of the gene coding for the first enzyme of agropine biosynthesis, the 3 end with that of the enzyme octopine synthase. The chimeric SPT gene has been linked to a selectable kanamycin resistance gene, and introduced into Nicotiana tabacum and Nicotiana plumbaginifolia by selection for the linked kanamycin resistance marker. Streptomycin resistance was expressed in some but not all of the kanamycin-resistant lines and was transmitted to the seed progeny as a dominant nuclear trait.     

Bleomycin resistance: a new dominant selectable marker for plant cell transformation

Summary Plant cells are sensitive to the antibiotic bleomycin, a DNA damaging glycopeptide. A bleomycin resistance determinant, located on transposon Tn5 and functional in bacteria, has been cloned in a plant expression vector and introduced into Nicotiana plumbaginifolia using Agrobacterium tumefaciens. The expression of this determinant in plant cells confers resistance to bleomycin and allows selection of transformed plant cells.     

Phleomycin resistance as a dominant selectable marker for plant cell transformation

Tobacco cells are sensitive to bleomycin and phleomycin. The Tn5 and the Streptoalloteichus hindustanus (Sh) bleomycin resistance (Ble) genes conferring resistance to these antibiotics have each been inserted into two plant expression vectors. They are flanked by the nopaline synthase (nos) or the cauliflower mosaic virus (CaMV) 35S promoters on one side, and by the nos polyadenylation signal on the other. These four chimaeric genes were introduced into the binary transformation vector pGA 492, which were thereafter mobilized into Agrobacterium tumefaciens strain LBA 4404. The resulting strains were used to transform Nicotiana tabacum cv. Xanthi nc using the leaf disc transformation procedure. In all cases, phleomycin- and bleomycin-resistant tobacco plants were regenerated from transformed cells under selective conditions; however the highest frequency of rooted plants was obtained when transformation was carried out with the Sh Ble gene under the control of the 35S promoter. Phleomycin resistance was stably transmitted to sexual offspring as a dominant nuclear trait as confirmed by Southern blotting.     

一种获取巴斯德毕赤酵母高拷贝重组子的新方法

目的：探讨一种简化的高效获取用于表达重组蛋白的巴斯德毕赤酵母高拷贝重组子的新方法,即多重转化筛选法。方法：按经典方法转化宿主菌和G418多拷贝筛选法从转化子中筛选出含目的的基因表达盒拷贝数最高的菌株,然后以之作为新一轮的转化宿主菌。如此重复进行数轮上述化筛选过程,直到获得含有尽可能多拷贝目的的基因表达盒的重组子;同时将此方法,即多重转化筛选法与经典方法进行比较。结果：与经典方法相比,多重转化筛选法通过筛选较少量转化子即可轻而易举地获得较多的高拷贝重组子,工作量更小,效率更高。结论：多重转化筛选法是一种更高效地获取毕赤酵母高拷贝重组子的新方法。     

2-Deoxyglucose resistance: a novel selection marker for plant transformation

A novel selection marker for plant transformation alternative to antibiotic and herbicide resistance is described. The selective agent applied is 2-deoxyglucose (2-DOG) which in the cytosol of plant cells is phosphorylated by hexokinase yielding 2-DOG-6-phosphate (2-DOG-6-P). 2-DOG-6-P exerts toxic effects on overall cellular metabolism leading to cell death. We observed that constitutive expression of the yeast DOG ^R1 gene encoding a 2-DOG-6-P phosphatase resulted in resistance towards 2-DOG in transgenic tobacco plants. This finding was exploited to develop a selection system during transformation of tobacco and potato plants. The lowest concentration of 2-DOG leading to nearly complete inhibition of regeneration of wild-type explants was found to range between 400 and 600 mg/l 2-DOG for tobacco, potato and tomato plants. After Agrobacterium tumefaciens-mediated transformation cells expressing the DOG ^R1 gene were selected by resistance to 2-DOG. More than 50% of tobacco explants formed shoots and on average 50% of these shoots harboured the DOG ^R1 gene. Similar results were obtained for potato cv. Solara. The acceptability of the resistance gene derived from baker's yeast, the unobjectionable toxicological data of 2-DOG as well as the normal phenotype of DOG ^R1-expressing plants support the use of this selection system in crop plant transformation.     

Genetic transformation of Trametes versicolor to phleomycin resistance with the dominant selectable marker shble

We have developed a stable, DNA-mediated transformation system for the white-rot basidiomycete Trametes versicolor based on the dominant selectable marker shble (phleomycin resistance). We employed a vector containing the selectable marker under control of expression sequences from the basidiomycete Schizophyllum commune and a polyethylene glycol/ CaCl2 protoplast-fusion technique to introduce the transforming DNA. This transformation system generated stable phleomycin-resistant transformants at a frequency of four to seven transformants/microg of transforming DNA.     

Multidrug resistance as a dominant molecular marker in transformation of wine yeast

Pure wine yeast cultures are increasingly used in winemaking to perform controlled fermentations and produce wine of reproducible quality. For the genetic manipulation of natural wine yeast strains dominant selective markers are obviously useful. Here we demonstrate the successful use of the mutated PDR3 gene as a dominant molecular marker for the selection of transformants of prototrophic wine yeast Saccharomyces cerevisiae. The selected transformants displayed a multidrug resistance phenotype that was resistant to strobilurin derivatives and azoles used to control pathogenic fungi in agriculture and medicine, respectively. Random amplification of DNA sequences and electrophoretic karyotyping of the host and transformed strains after microvinification experiments resulted in the same gel electrophoresis patterns. The chemical and sensory analysis of experimental wines proved that the used transformants preserved all their useful winemaking properties indicating that the pdr3-9 allele does not deteriorate the technological properties of the transformed wine yeast strain.     

mazF as a counter-selectable marker for unmarked genetic modification of Pichia pastoris

In this study, we demonstrate a novel method for unmarked genetic modification of the methylotrophic yeast Pichia pastoris , in which the Escherichia coli toxin gene mazF was used as a counter-selectable marker. mazF was placed under the tightly controlled AOX1 promoter, and the induced expression of MazF in P. pastoris halted cell growth. A modular plasmid was constructed in which mazF and a Zeocin resistance gene acted as counter-selectable and active-selectable markers, respectively, and the MazF-ZeoR cassette was flanked by two direct repeats for marker recycling. Linearized delivery vectors constructed from the modular plasmid were integrated into the P. pastoris genome via homologous recombination, introducing genetic modifications. Upon counter-selection with methanol medium, which induces the AOX1 promoter, the markers were recycled efficiently via homologous recombination between the direct repeats. We used this method successfully to knock-out the ARG1 and MET2 genes, knock-in a green fluorescent protein expression cassette, and perform site-directed mutagenesis on the ARG1 gene, all without introducing unwanted selection markers. The novel method allows repeated use of the selectable marker gene for multiple modifications and will be a useful tool for P. pastoris studies.     

A new dominant selectable marker for genetic transformation; Hsp70-opd

New P element plasmids containing the organophosphate-degrading gene opd as a dominant selectable marker were tested as transformation vectors in Drosophila melanogaster. One of these vectors was modified by the addition of the D. melanogaster mini-white gene as a comarker. When transformed individuals were identified using paraoxon selection for opd alone, results were similar to those obtained with mini-white. No false positives were recovered, however one strain contained the mini-white gene but inadequate resistance to survive our screening regimen due to a defective Hsp70-opd gene. Results suggest that Hsp70-opd is similar to mini-white for distinguishing transformed individuals, but does not require time-consuming individual examination. Due to the mode of action of organophosphorus nerve agents, Hsp70-opd has potential as a selectable marker in numerous animals beside fruit flies.     

Direct transformation of a clinical isolate of Candida parapsilosis using a dominant selection marker

Candida parapsilosis is a human pathogenic fungus with increasing importance, particularly in nosocomial infections. For detailed molecular genetic explorations of prototrophic clinical isolates of C. parapsilosis, we developed an efficient transformation system based on a dominant selectable marker. The gene encoding resistance to mycophenolic acid (MPA) was used for selection in yeast transformation. C. parapsilosis cells were transformed with a plasmid vector containing the Candida albicans inosine monophosphate dehydrogenase gene (IMH3) responsible for mycophenolic acid resistance. Transformation was carried out both by electroporation and by the lithium acetate (LiAc) method. The LiAc method resulted in very poor transformation efficiency, while the modified electroporation method yielded a high number of mitotically stable transformants exhibiting unambiguous MPA resistance. Two hundred transformants were analysed for the presence of the C. albicans IMH3(r) gene by polymerase chain reaction. Integration of single or multiple plasmid copies into the genomic DNA of C. parapsilosis was determined by Southern hybridization. To our knowledge, the present study is the first report about a method based on a dominant selectable marker for the transformation of a prototrophic, clinical isolate of C. parapsilosis. The described technique may prove to be an efficient tool for the examination of the biology and virulence of this pathogenic yeast.     

A new dominant hybrid selective marker for higher eukaryotic cells

A new dominant hybrid genetic marker, suitable for selection in higher eukaryotic cells, has been obtained by linking the promoter region of the Herpes simplex virus type I thymidine kinase gene to the gene coding for the aminoglycoside 3′ phosphotransferase coded for by the Tn5 transposon. As described for yeasts (Jimenez &; Davies, 1980), the expression of the enzyme allows the formation of cellular clones resistant to the antibiotic G-418, which is otherwise toxic to the cells. This dominant marker was expressed in all cell lines tested (human, simian and murine). The construction of the hybrid gene has been optimized and, for a given construction, transformation efficiency depends on the cell line. We also report that an unlinked gene can be cotransferred into cells with the marker and be expressed at high frequency.     

An improved integrative transformation system for Pichia pastoris with DNA-polyethylenimine-dextran sulfate nanoparticles

Pichia pastoris is a suitable host for selecting mutant enzymes, since it can secret many gene products to the medium. However, poor transformation efficiency is often encountered. This makes it difficult to obtain a large number of transformants necessary to thoroughly cover a large library. We report here that pre-coating DNA with polyethyleneimine and dextran sulfate increased gene integration significantly in Pichia electroporation. This improvement on integrative efficiency has been proved in different voltage, resistance, cell phase, and DNA concentrations. The condensed DNA nanoparticles make Pichia available as a host for screening large libraries of random mutants.     

Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants.

The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest.     

Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker

Reverse-genetic studies of chloroplast genes in the green alga Chlamydomonas reinhardtii have been hampered by the paucity of suitable selectable markers for chloroplast transformation. We have constructed a series of vectors for the targeted insertion and expression of foreign genes in the Chlamydomonas chloroplast genome. Using these vectors we have developed a novel selectable marker based on the bacterial gene aphA-6, which encodes an aminoglycoside phosphotransferase. The aphA-6 marker allows direct selection for transformants on medium containing either kanamycin or amikacin. The marker can be used to inactivate or modify specific chloroplast genes, and can be used as a reporter of gene expression. The availability of this marker now makes possible the serial transformation of the chloroplast genome of Chlamydomonas. Received: 26 October 1999 / Accepted: 28 December 1999     

Biolistic transformation of soybean using a new selectable marker gene conferring resistance to dinitroanilines

A biolistic transformation of soybean using the construct pAHTUAm, which contains a mutant α-tubulin gene, as a selectable marker gene that confers resistance to dinitroaniline herbicides, as well as the additional construct pAHTUB1, which carries a full-length barley β-tubulin gene that ensures correct co-expression of exogenous tubulin in cells of transgenic soybean lines, is carried out. It is established that 10 μM trifluralin is the most optimal selective concentration to pick up transformed soybean lines. The transgenic nature of the selected regenerants is confirmed by Southern blotting hybridization using a specific probe to the α-tubulin selectable gene     

Construction of a binary vector for transformation of Arabidopsis thaliana with a new selection marker

A new binary vector, pZT4B, containing the UDP-N-acetylglucosamine: dolichol phosphate N-acetylglucosamine-1-P transferase (GPT) gene as a selection marker, was constructed. The green fluorescent protein (GFP) gene was inserted into pZT4B, and the resulting plasmid was used in the transformation of Arabidopsis. All of six independent transformants obtained after selection with 0.3 mg/l tunicamycin contained the transgene and showed GFP fluorescence.     

New selectable marker/auxotrophic host strain combinations for molecular genetic manipulation of Pichia pastoris

We describe the isolation and characterization of three new biosynthetic genes-ARG4, ADE1, and URA3-from the methylotrophic yeast Pichia pastoris. The predicted products of the genes share significant sequence similarity to their Saccharomyces cerevisiae counterparts, namely argininosuccinate lyase, PR-aminoimidazolesuccinocarboxamide synthase, and orotidine-5'-phosphate decarboxylase, respectively. Along with the previously described HIS4 gene, each gene was incorporated as the yeast selectable marker into a set of shuttle vectors designed to express foreign genes in P. pastoris. In addition, we have constructed a series of host strains containing all possible combinations of ade1, arg4, his4, and ura3 auxotrophies to be used with these new vectors.     

利用Pichia pastoris生产S-腺苷甲硫氨酸的发酵工艺

在摇瓶中考察了重组Pichia pastoris发酵的诱导剂量,L-甲硫氨酸,以及pH对腺苷甲硫氨酸产量的影响.放大到3.7 L发酵罐和30 L发酵罐后,研究了重组细胞的发酵过程变化,对S-腺苷甲硫氨酸初步纯化.摇瓶中优化后的发酵条件是:每天添加1%甲醇诱导,L-甲硫氨酸为50mmol/L,培养基pH 5.0.培养144 h后SAM产量达到2.32 g/L.3.7 L发酵罐中发酵251 h后细胞浓度为120 g/L,SAM总量为15.18 g.放大到30 L发酵罐中,发酵225.5 h后细胞浓度约为120 g/L,SAM总量为145.05 g.纯化后SAM的纯度为93.5%,回收率为84.5%.