重组普鲁兰酶在枯草芽孢杆菌中的高效表达

普鲁兰酶可特异性地水解支链淀粉得到直链淀粉,因而在淀粉加工过程中具有重要的应用。本研究从Bacillus naganoensis ATCC53909基因组中克隆了普鲁兰酶基因pul,并克隆到大肠杆菌-枯草芽孢杆菌穿梭载体p BE中,构建表达载体p BE-pul。在此基础上,将来源于枯草芽孢杆菌、地衣芽孢杆菌以及解淀粉芽孢杆菌中的17个高表达基因的启动子分别克隆到表达载体p BE-pul中,并转化至Bacillus subtilis ATCC6051?10,成功构建了十七株含有不同启动子介导普鲁兰酶分泌表达的重组菌株。对重组菌株的分泌表达比较发现,启动子P43和Pspov G介导的普鲁兰酶活性明显优于其他启动子,其中Pspov G介导的普鲁兰酶活性更高。同时,还使用了启动子Pspov G介导N端的108个氨基酸缺失的pul324突变体进行分泌表达。通过对17种启动子的比较和两个普鲁兰酶基因的比较,本研究构建的一株重组菌株的普鲁兰酶的表达更为高效,其活性高达389.85 U/mL,后者显著高于现有的相关报道。     

An estradiol‐inducible promoter enables fast,graduated control of gene expression in fission yeast

The fission yeast Schizosaccharomyces pombe lacks a diverse toolkit of inducible promoters for experimental manipulation. Available inducible promoters suffer from slow induction kinetics, limited control of expression levels and/or a requirement for defined growth medium. In particular, no S. pombe inducible promoter systems exhibit a linear dose–response, which would allow expression to be tuned to specific levels. We have adapted a fast, orthogonal promoter system with a large dynamic range and a linear dose response, based on β‐estradiol‐regulated function of the human oestrogen receptor, for use in S. pombe. We show that this promoter system, termed Z₃EV, turns on quickly, can reach a maximal induction of 20‐fold, and exhibits a linear dose response over its entire induction range, with few off‐target effects. We demonstrate the utility of this system by regulating the mitotic inhibitor Wee1 to create a strain in which cell size is regulated by β‐estradiol concentration. This promoter system will be of great utility for experimentally regulating gene expression in fission yeast. Copyright © 2017 John Wiley & Sons, Ltd.     

A DNA microarray for fission yeast: minimal changes in global gene expression after temperature shift

Completion of the fission yeast genome sequence has opened up possibilities for post-genomic approaches. We have constructed a DNA microarray for genome-wide gene expression analysis in fission yeast. The microarray contains DNA fragments, PCR-amplified from a genomic DNA template, that represent > 99% of the 5000 or so annotated fission yeast genes, as well as a number of control sequences. The GenomePRIDE software used attempts to design similarly sized DNA fragments corresponding to gene regions within single exons, near the 3'-end of genes that lack homology to other fission yeast genes. To validate the design and utility of the array, we studied expression changes after a 2 h temperature shift from 25 degrees C to 36 degrees C, conditions widely used when studying temperature-sensitive mutants. Obligingly, the vast majority of genes do not change more than two-fold, supporting the widely held view that temperature-shift experiments specifically reveal phenotypes associated with temperature-sensitive mutants. However, we did identify a small group of genes that showed a reproducible change in expression. Importantly, most of these corresponded to previously characterized heat-shock genes, whose expression has been reported to change after more extreme temperature shifts than those used here. We conclude that the DNA microarray represents a useful resource for fission yeast researchers as well as the broader yeast community, since it will facilitate comparison with the distantly related budding yeast, Saccharomyces cerevisiae. To maximize the utility of this resource, the array and its component parts are fully described in On-line Supplementary Information and are also available commercially.     

Substrate-inducible versions of internal alternative NADH: ubiquinone oxidoreductase from Yarrowia lipolytica

In standard laboratory strains of the obligate aerobic yeast Yarrowia lipolytica, respiratory chain complex I (proton-translocating NADH : ubiquinone oxidoreductase) is an essential enzyme, since alternative NADH dehydrogenase activity is located exclusively at the external face of the mitochondrial inner membrane. Deletions and other loss-of-function mutations in genes for nuclear coded subunits of complex I can be obtained only when an internal version of the latter enzyme, termed NDH2i, is introduced. In contrast to recent findings with Neurospora crassa, external alternative NADH dehydrogenase activity is dispensable in complex I deletion strains of Y. lipolytica. We used regulable promoters to create strains which express internal alternative NADH dehydrogenase in a substrate-dependent manner. The ability to switch between complex I-dependent and -independent mode of growth simply by changing the carbon source is an important prerequisite for screens for both loss-of-function and inhibitor resistance mutation. The isocitrate lyase promoter (pICL1), in combination with a NDH2i allele that results in reduced expression and activity, was most promising. In the presence of complex I inhibitors, this construct allowed growth on acetate, but not on glucose minimal media. A somewhat higher background was observed with the acyl-CoA oxidase 2 (pPOX2) promoter on glucose minimal media.     

Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has proved to be an excellent model organism to study the function of proteins. One of the many advantages of yeast is the many genetic tools available to manipulate gene expression, but there are still limitations. To complement the many methods used to control gene expression in yeast, we have established a conditional gene deletion system by using the FLP/FRT system on yeast vectors to conditionally delete specific yeast genes. Expression of Flp recombinase, which is under the control of the GAL1 promoter, was induced by galactose, which in turn excised FRT sites flanked genes. The efficacy of this system was examined using the FRT site-flanked genes HSP104, URA3 and GFP. The pre-excision frequency of this system, which might be caused by the basal activity of the GAL1 promoter or by spontaneous recombination between FRT sites, was detected ca. 2% under the non-selecting condition. After inducing expression of Flp recombinase, the deletion efficiency achieved ca. 96% of cells in a population within 9 h. After conditional deletion of the specific gene, protein degradation and cell division then diluted out protein that was expressed from this gene prior to its excision. Most importantly, the specific protein to be deleted could be expressed under its own promoter, so that endogenous levels of protein expression were maintained prior to excision by the Flp recombinase. Therefore, this system provides a useful tool for the conditional deletion of genes in yeast.     

A series of yeast shuttle vectors for expression of cDNAs and other DNA sequences

Expression/shuttle vectors for the yeast Saccharomyces cerevisiae have usually been large plasmids with only one or a small number of sites that are suitable for cloning and expression. We report here the construction and properties of a series of 12 expression vectors with multiple (four to eight) unique sites in their polylinkers which allow directional cloning and expression of DNA sequences under four different promoters. Eleven of these plasmids replicate at high copy number in Escherichia coli, and all have the yeast TRP1 gene, and the 2 μm origin including REP3 sequence, allowing selection and high copy number replication in yeast. Six of the plasmids are designed for the construction and selection of cDNA libraries from various eukaryotic organisms, allowing directional cloning and expression of cDNAs. All of these six have similar polylinkers containing a unique promoter proximal EcoRI site and a unique promoter distal XhoI site, allowing for directional cloning and expression of ‘ZAP’-type cDNAs. cDNAs that complement a wide variety of yeast mutants can be selected from libraries constructed in this way. The four alternative promoters, ADH2, PGK, GAL10 and SV40 were compared for their relative activity, both in E. coli and in yeast. All yeast promoters showed substantial activity in E. coli with ADH2 showing the highest activity. ADH2 also was well-regulated in yeast, showing very high relative activity under derepressing conditions. cDNAs selected by genetic complementation from libraries constructed in these vectors should be easily subclonable into other vectors, allowing expression in different eukaryotic organisms, DNA sequencing or site-directed mutagenesis.     

Yeast vectors for the integration/expression of any sequence at the TYR1 locus

We have constructed new yeast vectors for targeted integration and conditional expression of any sequence at the Saccharomyces cerevisiae TYR1 locus which becomes disrupted. We show that vector integration is not neutral, causing prototrophy for tyrosine and auxotrophy for the vector's selectable marker (uracil or leucine, depending on the vector used). This feature allows a double screening of transformed yeast cells, improving the identification of colonies with the desired chromosomal structure. The GAL10 gene promoter has been added to drive conditional expression of cloned sequences. Using these vectors, chromosomal structure verification of recombinant clones is no longer necessary, since the noise of non-homologous recombination, as well as spontaneous reversion of the selected phenotype, can easily be identified. The ability of the vector to conditionally control gene expression has been confirmed using the gene for the green fluorescent protein (GFP) as a reporter.     

毕赤酵母组成型高效表达启动子的筛选鉴定

为获得高产量、高活性的葡萄糖氧化酶(GOD)酵母表达菌株,优化了GOD的T132S/T56V双突变编码序列,将之克隆到表达载体pGAP_k上得到表达载体pGAP_k-h₂-GOD,以pGAP启动GOD基因的表达。将pGAP_k-h₂-GOD上的pGAP启动子替换为pGCW14和pAOX1,并对pGCW14启动子进行改造,得到3种pGCW14的改造体,最终得到包括pGAP_k-h₂-GOD在内的6种不同启动子的重组表达载体。将这6种载体转化毕赤酵母GS115,建立了一种简便高效的筛选方法初步筛选出GOD重组菌株,再对筛选到的转化子进行发酵培养,测定发酵上清液的GOD酶活力,以此来比较各种启动子启动效率的强弱。结果显示:pGCW14的启动效率是pGAP的3~5倍,改造后的启动子pGCW14+G20A/C-467T(0.767)和pGCW14-UA(0.689)的启动效率较原始的pGCW14(0.574)都有明显的提高,尤其pGCW14+G20A/C-467T启动效率最高,比原始pGCW14提高了32.5%左右。与诱导型启动子pAOX1(1.187)相比,pGCW14+G20A/C-467T(1.109)的启动效率仅比其低6.6%左右。结论:改造后的启动子可望在毕赤酵母组成型高效表达的研究应用中具有一定的前景。     

One-step measurement of firefly luciferase activity in yeast

Firefly luciferase is often used as a sensitive genetic reporter in various cell types. The pitfall in yeast, however, has been the need to break down the rigid cells in order to measure the enzyme activity. In this study we have removed the peroxisomal targeting codons from the Photinus pyralis luciferase gene (luc) and shown that in the yeast Saccharomyces cerevisiae this modified luciferase gives high levels of light emission that is easy to measure from intact living cells. Furthermore, cells with the modified luciferase grew essentially faster than those with the wild-type luciferase, indicating that peroxisomal targeting of a foreign enzyme puts some constraints to cellular viability. As a model system we used two different reporter constructs. In the first, expression of the luciferase gene is under control of CUP1-promoter, a well known yeast promoter that is inducible by copper ions. In the second, luciferase activity is dependent on activation of the human oestrogen receptor and its interaction with oestrogen-responsive elements incorporated in a yeast promoter. The luciferase activity measurement could be done on a 96-well plate by simple addition of the substrate, D-luciferin, at a moderately acidic pH of 5.0. The ease of use of the non-peroxisomal luciferase makes it an interesting alternative for reporter genes that are conventionally used in yeast, such as lacZ.     

An improved tetO promoter replacement system for regulating the expression of yeast genes

Regulatable promoters are commonly used to control the expression of, especially, essential genes in a conditional manner. Integration of such promoters upstream of an ORF using one-step PCR-mediated homologous recombination should be particularly efficient. However, integration of the original KanMX4-tetO promoter cassette (Belli et al., 1998a) into the relatively short upstream regions of many yeast genes is often problematic, presumably due to the size (3.9 kb) of the replacement cassette. We have created a new, shorter, KanMX4-tetO cassette by removing the transactivator (tTA) sequence from the original cassette. The transactivator (tTA) has been integrated into the yeast genome to create a new strain for use with the new system, which has a greatly increased efficiency of promoter substitution. With it, we have been able to create strains that could not be made with the original cassette. To increase the throughput of promoter substitutions, we have developed a new assay for testing doxycycline sensitivity, based on liquid culture using microtitre trays. Altogether, the components of this new 'tool kit' greatly increase the efficiency of systematic promoter substitutions.     

Expression and in vivo determination of firefly luciferase as gene reporter in Saccharomyces cerevisiae

The LUC gene coding for Photinus pyralis firefly luciferase was cloned in different yeast episomal plasmids in order to assess its possibilities as an in vivo reporter gene. Activity of the enzyme in transformed cells in vivo was measured by following light emission and assay conditions optimized in intact cells, with regard to oxygen concentration, temperature, cell concentration in assay mixtures and external ATP concentration. Among the factors tested, light emission was drastically influenced by the external pH in the assay (which resulted in a ten-fold amplification signal) and by substrate permeability. The growth phase of the cells was also important for the level of activity detected. Cloning of firefly luciferase gene under the control of different yeast-regulated promoters (ADH1, GAL1–10) enabled us to measure their strength which correlated well with previously described data. We conclude that firefly luciferase is an adequate gene reporter for the in vivo sensitive determination of gene expression and promoter strength in yeast.     

Facilitating functional analysis of the Saccharomyces cerevisiae genome using an EGFP-based promoter library and flow cytometry

A promoter library was generated to facilitate identification of differentially regulated promoters in Saccharomyces cerevisiae. The library was constructed in a vector containing two reporter genes (EGFP and lacZ) divergently arranged about a unique cloning site. Approximately 2x10(5) clones were obtained and a flow cytometer was used to screen the library for copper-induced EGFP expression. A DNA fragment conferring copper-inducible expression of EGFP was rapidly identified. This DNA fragment, which contained several motifs associated with copper and oxidative stress homeostasis, lies upstream of two 'orphan' genes of unknown function. Further studies comparing expression from episomal vs. integrative vectors showed that construction of a similar library using an integrative vector would further enhance rapid identification of genes that are differentially regulated in S. cerevisiae. The ability to identify regulated promoters rapidly should facilitate the functional analysis of the yeast genome by identifying genes induced by specific physiological conditions.     

Screening for novel essential genes of Saccharomyces cerevisiae involved in protein secretion

We describe here a screening procedure devised for searching new genes involved in protein secretion in Saccharomyces cerevisiae. The screening procedure takes advantage of yeast strains constructed within the EUROFAN project, in which the promoters of the novel essential genes were replaced by the doxycycline-regulated tetO(7)-CYC1 promoter. This promoter is active in normal growth medium but results in downregulation of the gene in the presence of doxycycline. The yeast cells were grown in the presence or absence of doxycycline, and both the growth and secretion of the heat shock protein, Hsp150p, into the culture medium were determined. In seven strains there was a specific effect on protein secretion. In a strain in which the RPN5 gene was downregulated, the level of secreted Hsp150p was increased compared to the control culture. When RER2 was downregulated, cells secreted Hsp150p that was not of the mature size. In five strains, secretion was more severely reduced than cell growth. One of these downregulated genes, YGL098w, was recently reported to encode an ER-located t-SNARE, USE1. Four of the genes detected, NOG2, NOP15, RRP40 and SDA1, encode proteins involved in ribosome assembly, suggesting a possible new signalling pathway between ribosome biogenesis and production of secreted proteins. The results obtained here indicate that the present screen could be successfully used in larger scale to identify novel secretion-related genes.