瑞氏木霉纤维素酶基因在酿酒酵母中的表达研究

从瑞氏木霉（Trichoderma reesei）cDNA文库中克隆到外切葡聚糖纤维二糖水解酶Ⅰ（CBHI）eDNA基因，将该基因分别连接到酿酒酵母（Saccharomyces cerevisiae）表达载体pAJ401和pVT100_U上，构建了重组质粒pAJ401-cbhl和pVTIOO_U-cbhl。分别电转化2个重组质粒转移至酿酒酵母H158中，得到重组酵母HPC和HAC，实现了CBHI的分泌型表达。再将质粒pAJ401-cbhl转入已含有瑞氏木霉内切葡聚糖酶Ⅰ基因egl的重组酵母Hlm中，构建了同时表达cbhl和egl的重组酵母菌株HMEPC。比较HMEPC和Hlm对纤维素底物的降解，前者滤纸酶活比后者提高14．3％，表明CBHI与EGI对滤纸降解有协同作用。     

普鲁兰酶突变体文库高通量筛选方法的建立及应用

在摇瓶水平实现普鲁兰酶基因(Gen Bank Accession No:AX203843)在大肠杆菌BL21(DE3)的异源表达,并成功按规格缩小实现了基于深孔板的高通量细胞培养,同时建立了基于深孔板的普鲁兰酶酶活快速高效的高通量检测方法。利用易错PCR技术对普鲁兰酶基因进行定向进化,突变基因产物重组于表达载体p ET-28a(+)-pel B中,并导入大肠杆菌BL21(DE3)构建突变体文库,利用该高通量筛选方法实现了突变体文库的快速高效筛选并获得了一株突变菌株4A4,其表达量提高了46.5%。该方法不仅适用于淀粉酶、纤维素酶等酶类的高通量筛选,同时为菌株文库的筛选以及蛋白质的定向进化提供了一种新思路。     

树干毕赤酵母基因组文库的构建及纤维二糖酶基因的筛选

首次构建了树干毕赤酵母DNA基因组文库,该文库包含3 000个克隆,插入片段长度为0.5～8.0 kb。树干毕赤酵母染色体基因为15 441 179 bp,该文库覆盖1.08倍树干毕赤酵母染色体基因,筛选出任一基因或序列的概率为97%。提取文库中的质粒醋酸锂法转化酿酒酵母W303-1A,涂布于纤维二糖为唯一碳源的平板,通过纤维二糖酶对底物纤维二糖的底物特异性筛选文库,首次获得树干毕赤酵母来源的一个1.82 kb的纤维二糖酶基因。     

黑曲霉糖化酶基因在毕赤酵母中的克隆和表达

从高产糖化酶的黑曲霉的cDNA文库中筛选出糖化酶基因,并研究在毕赤酵母中的表达情况。运用RT-PCR从黑曲霉cDNA文库中克隆糖化酶基因的cDNA片段与载体pPIC9K相连,构建重组载体,电转化毕赤酵母GS115,筛选阳性克隆并进行研究。阳性克隆在MM培养基中发酵72 h和1%的甲醇的诱导的情况下,重组毕赤酵母产生的糖化酶酶活最大为15.6 U/mL。测定结果显示,其糖化酶大小为1 908 bp,编码636个氨基酸残基组成的蛋白质。经柱分离纯化其发酵上清液后,用SDS-PAGE电泳方法,测得分子质量大约为80 ku。黑曲霉糖化酶基因在毕赤酵母GS115中成功得到了表达。     

在酿酒酵母中重构棉子糖生物合成途径的基础研究

研究了在酿酒酵母中重构棉子糖生物合成途径,为后续高效生物合成棉子糖细胞工厂的构建奠定基础。敲除酿酒酵母中能降解棉子糖的蔗糖酶与α-半乳糖苷酶的基因构建出E1(Δsuc2::Δmel1);在此基础上,构建单基因表达拟南芥肌醇半乳糖合成酶基因gols1与gols3及棉子糖合成酶基因sip1与sip5的工程菌株,及构建双基因组合表达(gols1::sip1、gols1::sip5、gols3::sip1与gols3::sip5)工程菌株;比较工程菌株代谢产物如肌醇、UDP-半乳糖、肌醇半乳糖、蔗糖及棉子糖等生成情况,验证在酿酒酵母中重构棉子糖生物合成途径的可行性。研究表明,通过共表达外源肌醇半乳糖合成酶及棉子糖合成酶基因,并敲除能降解棉子糖的蔗糖酶与α-半乳糖苷酶的基因,在酿酒酵母中实现了棉子糖生物合成途径的重构;肌醇半乳糖合成酶与棉子糖合成酶基因的不同共表达组合,棉子糖生成量有差异;重构的棉子糖生物合成途经改变了酿酒酵母原始菌株的代谢流量。     

核盘菌诱导下甘蓝型油菜宁RS-1的SSH文库构建

以甘蓝型油菜宁RS-1为材料,用核盘菌（Sclerotinia sclerotiorum（Lib.）de Bary）对幼苗进行接种,提取经核盘菌诱导前和诱导后的宁RS-1叶片mRNA,经反转录成cDNA,以及经过酶解后加接头和两次杂交,构建差异表达的抑制消减（SSH）cDNA文库。正向消减文库是以诱导后的宁RS-1 cDNA为tester,以正常的宁RS-1 cD-NA为driver。所构建的正向文库重组率达到96.2%,反向文库重组率达到94.5%,文库容量均达到了2 190和2 780,表明所构建的文库质量良好。对从文库中随机挑取的克隆进行测序分析,获得了与抗病和代谢有关的基因,如γ-谷氨酰半胱氨酸酶基因、抗真菌蛋白基因、铁蛋白基因、ACC氧化酶基因等,为开展油菜抗菌核病重要功能基因的克隆和鉴定奠定了基础。     

抗克伦特罗核糖体展示单链抗体库的构建及鉴定

构建大容量核糖体展示抗克伦特罗单链抗体(single-chain fragment variant,scFv)文库,为进一步筛选抗体奠定基础。用克伦特罗人工抗原免疫小鼠,分离其脾细胞;Trizol法抽提总RNA,反转录成cDNA,PCR及重叠延伸PCR法构建核糖体展示scFv 文库。再通过PCR和重叠延伸PCR在scFv文库序列5＇端添加T7启动子、茎环结构、核糖体结合位点,3＇端添加间隔区序列(T20-V109)、茎环结构构建核糖体展示抗克伦特罗scFv文库。核糖体展示scFv文库与Easy T3载体连接,转化大肠杆菌DH5α感受态细胞,经蓝白斑筛选,挑取5个阳性克隆双酶切和测序鉴定。结果表明：用于建库材料的免疫小鼠效价为1:128000;成功扩增重链可变区基因大约为360bp和轻链可变区基因大约为330pb;核糖体展示scFv文库基因大约1200bp;库容量达1.75×1013;阳性重组质粒均含有1200bp插入片段;利用IMGT/V-QUEST数据库对单链抗体可变区进行序列分析结果表明,重链可变区和轻链可变区基因均由胚系基因重排得到,且与鼠源胚系基因同源率都达84%以上;氨基酸序列比对结果表明,重链可变区氨基酸序列同源率为37.82%,轻链可变区氨基酸序列同源率为39.09%。其中多样性主要发生在VH-CDR3、VL-CDR1、VL-CDR2和VL-CDR3。成功构建了大容量抗克伦特罗单链抗体核糖体展示文库。     

基于生物量产率的低产乙醇酿酒酵母的筛选

目的:建立一种从酿酒酵母突变库中快速筛选低产乙醇酵母菌株的方法。方法:以转录因子spt15随机突变酿酒酵母文库为研究模型,以高生物量产率作为低产乙醇酿酒酵母的筛选标记,利用24孔板对spt15突变酵母文库进行初筛。对初筛获得的目标菌株,以乙醇产量作为筛选标记进行复筛,最终获得乙醇合成能力较低且生物量较高的酿酒酵母菌株。结果:(1)利用易错PCR技术构建了spt15随机突变文库,并将其转化至酿酒酵母YS59,获得酿酒酵母YS59 spt15突变文库;(2)经初筛,获得14株生物量产率≥70%的酿酒酵母YS59 spt15突变菌株;(3)通过复筛,筛选出编号712的低产乙醇突变菌株,其乙醇合成量降低了28.1%;(4)通过序列比对,发现spt15-712上有9个碱基发生突变,分别是A161G,T426C,T462C,T493A,A506G,T546C,T622C,T658C,A750T9。结论:成功建立了一种高通量筛选低产乙醇酿酒酵母的方法 ,并利用这种方法筛选出乙醇合成量降低28.1%的低产乙醇突变菌株。     

白藜芦醇在酿酒酵母中的组合表达

将克隆自拟南芥的4cl基因和巨峰葡萄的rs基因,利用降落重叠延伸PCR的方法,成功构建了含有G418抗性筛选标记的酵母表达载体pRS42K-4CL以及含有潮霉素抗性筛选标记的酵母表达载体pRS42H-RS。采用LiAc/SS carrier DNA/PEG法将含有目的基因的2个载体共同转化至酿酒酵母工业菌株EC1118中,通过PCR及酶切鉴定等方法验证重组工程菌。以对香豆酸为底物,将获得的酿酒酵母工程菌于YPD液体培养基中发酵(25℃,150 r/min,96 h),发酵液用乙酸乙酯抽提后采用高效液相色谱(HPLC)法进行检测,其结果显示发酵产物中白藜芦醇的含量为0.78 mg/L。这表明,拟南芥的4cl基因与巨峰葡萄的rs基因在酿酒酵母工程菌中成功得到了表达,并且表达产物利用对香豆酸为前体物质合成了目标产物白藜芦醇。该研究为进一步实现白藜芦醇在酵母中的工业化生产奠定了基础。     

烟草优质品种叶片全长cDNA文库的构建和质量分析

为研究优质烟叶的分子基础,以翠碧一号不同生长时期的叶片为材料,CTAB法提取总RNA,利用SMART技术合成全长cDNA,之后按经修改的Clontech技术成功构建了烟草叶片全长cDNA文库.经鉴定,初级文库库容为3.85×106个克隆,重组率为100％,重组子插入片段集中在750-2000 bp之间.随机挑取25个克隆测序后,经生物信息学分析表明,烟草上已经报道的基因占10条,11条为全长基因序列;20条序列有功能注释.综合分析表明,所构建的文库质量较高,达到了基因的分离、筛选和克隆的建库目的.     

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.     

Isolation of five laccase gene sequences from the white-rot fungus Trametes sanguinea by PCR, and cloning, characterization and expression of the laccase cDNA in yeasts

To obtain laccase-gene-specific sequences from the white-rot fungus Trametes sanguinea M85-2, a PCR screening method was used. Degenerate primers were designed based on highly conserved copper-binding regions I and IV of known laccases and used to amplify laccase sequences from T. sanguinea M85-2 genomic DNA. A single 1.6-kbp DNA band was amplified and cloned into a vector. Partial sequences of 21 clones were classified into five groups (lcc1-5) and the deduced amino acid sequences were all homologous to known laccase sequences. Based on the partial sequence of lcc1, the 5'-end of its cDNA was obtained by a PCR termed 5' rapid amplification of cDNA ends (5'-RACE), and RT-PCR was then carried out using the 5'-primer and the poly-dT primer to obtain the full-length lcc1 cDNA. The obtained cDNA encoded a protein consisting of 518 amino acid residues and its first 21 amino acid residues were predicted to be the signal peptide for secretion. The conserved characteristic structures of laccase, such as copper-binding ligands, N-glycosylation sites, and cysteine residues for disulfide bridges, were observed. The genomic DNA sequence of the lcc1 gene was also cloned by PCR method and the sequence revealed 10 introns. The lcc1 cDNA was inserted into yeast vectors for heterologous expression by Saccharomyces cerevisiae and Pichia pastoris. Phenol-oxidizing activity was detected from transformants of the yeasts, indicating that the obtained cDNA encodes a laccase. Previously, two laccase isozymes were biochemically characterized and purified from T. sanguinea M85-2. Using the sequential PCR method presented here, we have obtained partial sequences of at least five laccase genes and one cDNA clone encoding a protein with laccase activity but without any enzymatic information, suggesting that expressed enzymes under restricted conditions may not represent all the isozymes in target microorganisms. PCR cloning and heterologous expression of the cloned genes can be an alternative method of screening enzymes if these enzymes have conserved sequences.     

Cloning and characterization of the lactate-specific inducible gene KlCYB2, encoding the cytochrome b(2) of Kluyveromyces lactis

In yeast the utilization of lactate requires two enzymes, the D and L-lactate ferricytochrome c oxidoreductase (D and L-LCR), which stereospecifically oxidize D- and L-lactate to pyruvate. These enzymes are nuclearly encoded and localized in mitochondria. In the yeast Kluyveromyces lactis, a mutant devoid of D- and L-LCR activities and unable to grow on racemic lactate was isolated. Transformation of the mutant with a K. lactis genomic library allowed the isolation of the KlCYB2 gene, restoring the growth on lactate and the L-LCR activity. The KlCYB2 gene and its flanking regions were sequenced (Accession No. AJ243324; EMBL/GenBank databases). The deduced amino acid sequence is highly homologous to the corresponding Saccharomyces cerevisiae and Hansenula anomala protein sequences previously characterized. The homology is missed in the N-terminal region, corresponding to the presequence cleaved during import into mitochondria. Analysis of KlCYB2 gene expression indicated that, in contrast to S. cerevisiae, the major regulatory feature is induction by lactate.     

Isolation of GCR1, a major transcription factor of glycolytic genes in Saccharomyces cerevisiae, from Kluyveromyces lactis

To study the function of GCR1, a gene involved in the expression of glycolytic genes in Saccharomyces cerevisiae, a Kluyveromyces lactis gene that complements the growth defect of a S. cerevisiae Deltagcr1 mutant was isolated. Introduction of this gene into the Deltagcr1 mutant also restored the activities of glycolytic enzymes. DNA sequencing of KlGCR1 predicted an open reading frame of a 767 amino acid protein with an overall identity of 33% and similarity of 48% to Gcr1p from S. cerevisiae. Its DDBJ/EMBL/GenBank Accession No. is AB046391.     

Efficient PCR-based gene disruption in Saccharomyces strains using intergenic primers

Gene disruptions are a vital tool for understanding Saccharomyces cerevisiae gene function. An arrayed library of gene disruption strains has been produced by a consortium of yeast laboratories; however their use is limited to a single genetic background. Since the yeast research community works with several different strain backgrounds, disruption libraries in other common laboratory strains are desirable. We have developed simple PCR-based methods that allow transfer of gene disruptions from the S288C-derived strain library into any Saccharomyces strain. One method transfers the unique sequence tags that flank each of the disrupted genes and replaces the kanamycin resistance marker with a recyclable URA3 gene from Kluyveromyces lactis. All gene-specific PCR amplifications for this method are performed using a pre-existing set of primers that are commercially available. We have also extended this PCR technique to develop a second general gene disruption method suitable for any transformable strain of Saccharomyces.     

Regulation of the expression of endopolygalacturonase gene PGU1 in Saccharomyces

Previous work in our laboratory has shown that Saccharomyces bayanus strain SCPP is the only reported yeast expressing the three types of pectolytic enzymes: pectin esterases, pectin lyases and polygalacturonases. One of these enzymes, the endopolygalacturonase (endoPG), hydrolyses plant-specific polysaccharide pectin. The endoPG encoding gene (PGU1) is also present in Saccharomyces cerevisiae. It has been shown that this endoPG is required for the development of pseudohyphae. Using genomic DNA, the PGU1-1 and PGU1-2 promoters of these strains have been amplified and used to construct gene fusions with the beta-galactosidase gene. On the basis of beta-galactosidase measurements, we compared the expression of both promoters in different environmental conditions in order to identify their modulation. We have shown that the PGU1 gene is upregulated by the presence of the pectin and the product resulting from endopolygalacturonase activity. Moreover, expression of the PGU1 is also enhanced under respiratory and filament formation conditions.     

A universal assay for screening expression libraries for carbohydrases

Although many assays are available for the screening of expression libraries for carbohydrases, some enzymes cannot be detected because their substrates are incompatible with the existing assays. One thing that all carbohydrases have in common is that they increase the number of reducing ends when degrading their substrates. In this paper we explore the possibility of detecting this increase with the highly sensitive bicinchoninic acid (BCA) reducing value assay. This assay can be used for the detection of all carbohydrases degrading any polysaccharide; enzymes with either an exo- or an endo-type of mechanism can be detected at the same time. A cDNA library of Aspergillus tubigensis expressed in Kluyveromyces lactis clones, was screened with this assay for the presence of xylogalacturonan degrading enzyme(s). High background absorbances caused by culture medium, by proteins produced by the clones and by substrate could be dealt with by using the precautions described in this note. Three xylogalacturonase producing clones were found using this procedure.     

α-乙酰乳酸脱羧酶基因在酿酒酵母中稳定表达的策略

综述了宿主细胞的表达特点，外源基因的来源，表达载体和转化方法对α-乙酰乳酸脱羧酶基因在酿酒酵母中稳定表达的影响，并在此基础上提出了相应的策略。     

A single-copy suppressor of the Saccharomyces cerevisae late-mitotic mutants cdc15 and dbf2 is encoded by the Candida albicans CDC14 gene.

The Saccharomyces cerevisiae CDC15, DBF2, TEM1 and CDC14 genes encode regulatory proteins that play a crucial role in the latest stages of the M phase of the cell cycle. By complementation of a S. cerevisiae cdc15-lyt1 mutant with a Candida albicans centromeric-based genomic library, we have isolated a homologue of the protein phosphatase-encoding gene CDC14. The sequence analysis of the C. albicans CDC14 gene reveals a putative open reading frame of 1626 base pairs interrupted by an intron located close to the 5' region. Analysis of C. albicans cDNA proved that the intron is processed in vivo. The CaCDC14 gene shares 49% of amino acid sequence identity with the S. cerevisiae CDC14 gene, 46% with Schizosaccharomyces pombe homologue, 35% with Caenorhabditis elegans and 37% and 38% with human CDC14A and CDC14B genes, respectively. As expected, the C. albicans CDC14 gene complemented a S. cerevisiae cdc14-1 mutant. We found that this gene was able to efficiently suppress not only a S.cerevisiae cdc15-lyt1 mutant but also a dbf2-2 mutant in a low number of copies and allowed growth, although very slightly, of a tem1 deletant. Overexpression of the human CDC14A and CDC14B genes complemented, although very poorly, S. cerevisiae cdc15-lyt1 and dbf2-2 mutants, suggesting a conserved function of these genes throughout phylogeny. The sequence of CaCDC14 was deposited in the EMBL database under Accession No. AJ243449.     

A genetic screen for yeast genes induced by sustained osmotic stress

The budding yeast, Saccharomyces cerevisiae, responds to changes in external osmolarity through the activation of an osmosensing signal transduction pathway. Using lacZ-reporter gene fusions, clonal cell lines were screened for levels of beta-galactosidase activity in the presence or absence of osmotic stress. A screen of 9,000 transformants displayed 663 (7%) gene fusions that were active in rich medium. Each of the transformants were also assayed for gene activity 24 h following a transfer to high osmolarity medium (0.6 M NaCl) and of the 9,000 clonal cell lines, 86 (1%) displayed a decrease in expression, and seven (0.1%) displayed a reproducible increase in gene expression during primary screening. The chromosomal loci of the lacZ insertions were determined, and the gene(s) associated with that site was examined for osmotically induced expression using RNA blot analysis. Five stress-activated genes were analysed by RNA blot: YDL222C, NMD2, PTC7, FAA4 and YRF1. The genes identified by this screen encompass cellular adaptations to stress including signal transduction, protein myristoylation and fatty acid/sphingolipid content in the cell membrane.