热带假丝酵母ctpxa1基因缺失菌的构建及对长链二元酸积累的影响

Pxa1p为酿酒酵母Saccharomyces cerevisiae过氧化物酶体上的膜蛋白,与Pxa2p组成二聚体,参与转运长链脂肪酸进入过氧化物酶体过程。热带假丝酵母能够发酵烷烃和脂肪酸生产长链二元酸,而过氧化物酶体中发生的β-氧化会消耗产生的长链二元酸造成产率降低。本研究以热带假丝酵母Candida tropicalis 1798为宿主菌,通过基于PCR片段的同源单交换法,快速构建ctpxa1基因敲除菌株C.tropicalis 1798-pxa1。利用半定量RT-PCR技术,检测ctpxa1基因在C.tropicalis 1798、C.tropicalis 1798-pxa1的表达量,灰度值比值为2.03,表明ctpxa1在C.tropicalis 1798-pxa1中的表达被弱化。经144 h发酵,C.tropicalis 1798-pxa1比C.tropicalis 1798的十二碳二元酸产量明显提升,其产出浓度为10.3 g/L,比野生型菌株C.tropicalis 1798提高了94.3%。     

新一代高产甘油重组菌的构建及其初步发酵

产甘油假丝酵母(Candida glycerinogenes WL2002-5)是一株发酵生产甘油的工业化菌株。为进一步提高其产甘油能力,本研究利用前期研究中成功克隆的产甘油假丝酵母中甘油合成关键酶3-磷酸甘油脱氢酶基因CgGPD1,构建根癌农杆菌双元载体pCAM3300-zeocin-CgGPD1后,电击转化根癌农杆菌LBA4404,通过根癌农杆菌介导法(ATMT)转化产甘油假丝酵母,构建了产甘油假丝酵母重组菌。并从中筛选出一株酶活力和产甘油性能较好的产甘油假丝酵母重组菌株C.g-G8。以葡萄糖为底物摇瓶发酵96h后,重组菌C.g-G8的甘油产量比野生型菌株Candida glycerinogene提高18.06%,平均耗糖速率提高12.97%,平均酶活力提高27.55%。本研究成功利用ATMT法转化产甘油假丝酵母构建新一代高产甘油菌株。     

利用可重复使用的URA3标记基因建立热带假丝酵母基因敲除系统

热带假丝酵母(Candida tropicalis)在发酵工业中具有重要的应用潜力,但二倍体遗传结构和较低的遗传转化效率限制了其代谢工程育种技术的应用。建立可靠的遗传转化技术并高效的删除目的基因是代谢工程改造热带假丝酵母的重要前提。文章以C. tropicalis ATCC 20336为出发菌株,通过化学诱变筛选获得了尿嘧啶缺陷型突变株C. tropicalis XZX(ura3/ura3)。以丙酮酸脱羧酶(Pyruvate decarboxylase,PDC)基因作为靶基因构建了两端包含同源臂并在选择性标记C. tropicalis URA3(Orotidine-5′-phosphate decarboxylase,乳清酸核苷-5-磷酸脱羧酶)基因两侧同向插入源于沙门氏菌(Salmonella typhimurium)的hisG序列的基因敲除盒PDC1-hisG-URA3-hisG- PDC1(PHUHP),并转化宿主菌株C. tropicalis XZX,筛选获得PHUHP片段正确整合到染色体的PDC基因位点的转化子XZX02。在此基础上,将转化子XZX02涂布于5-FOA(5-氟乳清酸)选择培养基上,筛选得到URA3基因从PHUHP片段中丢失的营养缺陷型菌株XZX03。进一步构建了第2个PDC等位基因的删除表达盒PDCm- URA3-PDCm,并转化C. tropicalis XZX03菌株,获得转化子C. tropicalis XZX04。经PCR和DNA测序确认转化子C. tropicalis XZX04细胞染色体上的两个PDC等位基因被成功敲除。文章建立了一种营养缺陷型标记可重复使用的热带假丝酵母遗传转化技术,利用该技术成功敲除了细胞的PDC基因,为进一步利用代谢工程改造热带假丝酵母奠定了基础。     

脂肪醇氧化酶基因缺失对热带假丝酵母生理功能的影响

为考察热带假丝酵母(Candida tropicalis)脂肪醇氧化酶(FAO)基因缺失对菌株自身的影响,利用同源重组的方法敲除或回补FAO1和FAO2基因,研究突变株的生长情况和胞内脂肪醇氧化酶活性变化,并进-步评价细胞利用烷烃合成脂肪醇的能力.结果表明:成功构建了基因缺失突变株FTYT(ΔFAO11/ΔFAO12)...     

一种适用于产朊假丝酵母的基因敲除系统及其在gsh1基因敲除中的应用

报道一种适用于产朊假丝酵母Candida utilis的基因敲除系统,利用该敲除系统获得gsh1基因敲除杂合突变株。根据不同种属酵母菌γ-谷氨酰半胱氨酸合成酶(γ-GCS)蛋白质的保守序列,克隆C.utilis SZU 07-01的gsh1基因;以商品化质粒pPICZalpha A为基础,构建gsh1基因的敲除载体pPICZalpha A-kan 3,其中,kan基因的启动子TEF被替换为来自于C.utilis SZU 07-01的GAP启动子(pGAP:kan)。质粒电转化C.utilis,获得gsh1基因敲除杂合突变株C.utilis GSH-6。结合发酵培养得到的数据进行分析,突变株的γ-GCS酶活比出发菌株降低17.5%,GSH合成量降低61%,细胞干重降低18.5%。所构建敲除组件pGAP:kan的成功应用为从分子水平研究C.utilis中谷胱甘肽(GSH)的生理功能提供了一种新借鉴。     

能源上的应用

900841 木糖的同步发酵和异构化[英] 利用凝结芽孢杆菌(Bacillus coagulans)细胞的市售固定化木糖异构酶,通过将蔗糖转化为木酮糖,从而用木糖制备出乙醇。采用嫌气发酵可同步将木酮糖转化乙醇,所用酵母菌株有热带假丝酵母(Candida tropicalis)ATCC 9968、Y-11860和Y-1552、休哈塔假丝酵母(C.     

代谢工程改善野生酵母利用木糖产乙醇的性能

从256个自然样品中筛选得到1株可高效转化D-木糖的酵母。通过生理生化和分子生物学方法鉴定, 证实该菌株是属于Candida tropicalis。以该酵母为研究对象, 增加木糖醇脱氢酶表达量, 通过改变代谢流以达到提高酒精产率的目的。以pXY212-XYL2质粒为基础载体, 构建了含有潮霉素抗性的pYX212-XYL2-Hygro, 电击转化进入野生型C. tropicalis, 潮霉素抗性筛选, 得到含高拷贝木糖醇脱氢酶基因的重组菌株C. tropicalis XYL2-7。重组菌的比酶活达到0.5 u/mg protein, 比原始菌株提高了3倍。实验表明, 重组菌木糖醇得率比原始菌株降低了3倍, 酒精得率提高了5倍。首次通过实验验证了热带假丝酵母利用木糖产乙醇的可行性, 这对研究酵母利用秸秆、麦糠、谷壳等纤维质农业废弃物生产燃料乙醇具有重要启示。     

代谢工程改善野生酵母利用木糖产乙醇的性能

从256个自然样品中筛选得到1株可高效转化D-木糖的酵母。通过生理生化和分子生物学方法鉴定, 证实该菌株是属于Candida tropicalis。以该酵母为研究对象, 增加木糖醇脱氢酶表达量, 通过改变代谢流以达到提高酒精产率的目的。以pXY212-XYL2质粒为基础载体, 构建了含有潮霉素抗性的pYX212-XYL2-Hygro, 电击转化进入野生型C. tropicalis, 潮霉素抗性筛选, 得到含高拷贝木糖醇脱氢酶基因的重组菌株C. tropicalis XYL2-7。重组菌的比酶活达到0.5 u/mg protein, 比原始菌株提高了3倍。实验表明, 重组菌木糖醇得率比原始菌株降低了3倍, 酒精得率提高了5倍。首次通过实验验证了热带假丝酵母利用木糖产乙醇的可行性, 这对研究酵母利用秸秆、麦糠、谷壳等纤维质农业废弃物生产燃料乙醇具有重要启示。     

用毕赤酵母组成型分泌表达Canstatin-N

目的:用毕赤酵母的GAP启动子调控组成型表达Canstatin-N。方法:将canstatin-N基因重组于毕赤酵母表达载体pGAP9K的多克隆位点获得pGAP9K-can-N。用电转法将pGAP9K-can-N转化毕赤酵母GS115。筛选高G418抗性的克隆作为工程菌GS115(pGAP9K-can-N)。发酵GS115(pGAP9K-can-N)分泌表达Canstatin-N,用离子交换法纯化目标蛋白。结果:以葡萄糖为碳源,发酵48h分泌表达人血管能抑素蛋白56 mg/L。结论:用毕赤酵母的GAP启动子调控组成型表达的人血管能抑素蛋白具有诱发血管内皮细胞凋亡的生物活性。     

热带假丝酵母代谢烷烃过程中的β-氧化和代谢调控

热带假丝酵母(Candida tropicalis)能利用烷烃作唯一碳源和能源.当以烷烃或脂肪酸为碳源时,在细胞内可形成大量的过氧化物酶体(peroxisome),同时诱导生成脂肪酸β-氧化酶系,当以葡萄糖为碳源时,则极少有过氧化物酶体形成[1],一些C.tropicalis能氧化烷烃生成长链二元酸(long chain dicarboxylicacid,DCA).由于这些特征,人们从酶学、分子生物学和实际应用等方面对这种酵母进行了深入研究,并阐述了C.tropicalis代谢烷烃的途径、脂肪酸β-氧化酶系的生理功能及其几种重要酶的基因结构和酶活性的调控,阐明了它与哺乳动物细胞在脂肪酸代谢及调控方面上的差异;通过C.tropicalis突变株的筛选和发酵工艺的优化,使长链二元酸发酵技术实现了产业化[2,3].     

Susceptibility assays of Candida tropicalis to miconazole

In vitro evaluation of antifungal activity of imidazole derivatives is made difficult by the inhibitory effects of several factors such as inoculum size, growth form of the fungus, incubation temperature, the presence of complex substances, including divalent ions, which strongly influence final results. This is particularly evident when testing clinical isolates of C. tropicalis strains resistant to imidazole drugs. Our data based on assays of miconazole nitrate and miconazole sulfosalicylate against C. tropicalis show that it is possible to abolish various interference activities on the antimicrobial activity by suitable modifications of some cultural conditions. Thus, a study has been carried out to assess miconazole sulfosalicylate activity on C. tropicalis throughout experiments performed by contact test and agar diffusion test. The use of these techniques made it possible to display some activity of the imidazoles even against strains of C. tropicalis, which were defined as resistant using usual susceptibility assay conditions. Experimental conditions which cause the increase of susceptibility of C. tropicalis are related to factors that modify the barrier function and cellular permeability as demonstrated mainly by the effect of electrical conductivity (E.C.), pH of the medium and pretreatment of fungal inoculum with sodium dioctylsulfosuccinate (SDSS). Our results suggest that the correlation between drug dosage and inhibitory activity in vitro can be improved by the modifications proposed in this paper.     

Use of transmissible plasmids as cloning vectors in Caulobacter crescentus

Cloning vectors for studies of Caulobacter crescentus genes should be transferable between Escherichia coli and C. crescentus since a transformation system has not been developed for C. crescentus. We have tested a large number of vectors containing IncP or IncQ replicons and found that many of the vectors containing IncQ replicons, and all but one of the vectors containing IncP replicons, are readily transferred by conjugation into C. crescentus. All of the plasmids tested were maintained in C. crescentus at 1 to 5 copies per cell, but plasmids containing IncP replicons were more stable than plasmids containing IncQ replicons. Further studies with a derivative of the IncQ plasmid R300B showed that when a promoterless kanamycin (Km)-resistance gene (npt2) was inserted into the intercistronic region of the sul-aphC (Su^R-Sm^R) operon, Km resistance was expressed only when the npt2 gene was inserted such that it would be transcribed from the sul promoter. These data indicate that R300B does not contain sequences which would provide promoter function in C. crescentus in the orientation opposite to that of the sul operon and that any genes cloned in this orientation would require native promoters for expression. To provide greater versatility for cloning into R300B, additional vectors were constructed by the addition of multiple cloning sites in the intercistronic region of the sul-aphC operon. In addition, chromosomal DNA libraries were constructed in R300B and in the cosmid vector pLAFR1-7. Specific clones from these libraries containing genes of interest were identified by complementation of the appropriate C. crescentus mutants.     

Identification and characterization of Clostridium difficile promoter element that is functional in Escherichia coli

The promoter element involved in the expression of a previously characterized cloned clostridial antigen was isolated and characterized. A restriction fragment containing the promoter element of the Clostridium difficile insert was cloned using the promoter probe vector, pGA46. Subclones of the clostridial DNA insert in pGA46 were then analyzed by nucleotide sequencing and by S1 nuclease experiments. The clostridial promoter element exhibits a high degree of homology with typical Escherichia coli promoter elements. This sequence probably represents a unique class of clostridial promoter elements which, given their ability to function in E. coli and C. difficile, can be used in the construction of a shuttle vector capable of gene expression in E. coli and C. difficile.     

Gassericin T基因的合成及其组成型表达载体的构建

目的:构建Gassericin T基因毕赤酵母(Pichia pastoris)组成型表达载体。方法:根据乳酸菌素Gassericin T的基因序列,把Gassericin T的结构基因gatA编码的氨基酸的密码子转换成P.pastoris偏爱的形式,设计了6条59nt的寡聚核苷酸引物,通过3次连续PCR反应,获得了250bp左右的gat A片段(简称gat基因)。应用PCR方法从P.pastoris染色体中扩增了GAP启动子,大小为500bp左右,以其取代诱导型表达载体pPIC9K上的pAOX1,构建了组成型表达载体pGAP9K。将合成的gat基因克隆到pGAP9K质粒的多克隆位点中。结果:获得的gat及gap基因与预期结果一致,序列无碱基突变,构建的表达载体pGAP9K-gat经PCR、酶切鉴定完全正确。结论:成功构建了Gassericin T基因P.pastoris组成型表达载体,为下一步高效表达Gassericin T蛋白,进一步研究其作用机理及应用价值打下基础。     

Constitutive expression of barley α-amylase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by high-density cell culture

α-amy gene amplified from barley genome was cloned into MCS of pGAP9K to generate pGAP9K-α-amy which was then transformed into Pichia pastoris GS115 by electroporation. Transformants with multi-copies and high expression for the foreign gene were selected on G418 containing plate and expression analysis. The fermentation was carried out in a 50 l bioreactor with 20 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol-0.8% PTM4 to the growing culture for 54 h at 30°C. Under the control of GAP promoter (pGAP), α-amy gene was constitutively expressed. At the end of the fermentation, the α-AMY expression reached 125 mg/l, while the biomass growth was 186 as measured by absorption of 600 nm. The secreted α-AMY was purified to 97.5% by SP-Sepharose FF ion-exchange chromatography and affinity purification. The recombinant α-AMY showed activity on hydrolysis of starch.     

组蛋白去乙酰化酶CfSnt2调控果生刺盘孢生长发育和致病力

炭疽病是油茶Camellia oleifera的重要病害,该病害的优势致病菌是刺盘孢属Colletotrichum的果生刺盘孢C. fructicola,在全国的油茶产区普遍发生。我们前期发现组蛋白乙酰转移酶CfGcn5调控油茶果生刺盘孢的生长发育和致病过程,但组蛋白去乙酰化酶在该病菌中的生物学功能尚不清楚。本研究以组蛋白去乙酰化酶CfSnt2为研究对象,利用反向遗传学的方法,通过对野生型、CfSNT2基因敲除突变体及互补菌株的生物学表型进行比较分析,发现CfSNT2基因敲除突变体的菌丝生长速率明显减缓、分生孢子的产量显著减少、附着胞形成率降低、对细胞壁胁迫剂的响应异常,同时对油茶致病力显著减弱。以上现象说明CfSnt2调控果生刺盘孢的生长、产孢、附着胞的形成、对细胞壁完整性胁迫剂的耐受性及致病力。     

用GAP启动子在毕节酵母中组成型表达人血管抑制素

为探索用GAP启动子(PGAP)取代AOX1启动子(PAOX1),在毕节酵母(P．pastortis)中组成型表达外源蛋白的可能性,应用PCR方法从P．pastoris染色体中扩增了GAP启动子,以其取代诱导型表达载体pPIC9K上的PAOX1,构建了组成型表达载体pGAP9K。将人血管抑制素(AS)基因重组于pGAP9K的多克隆位点,获得含As基因的重组质粒pGAP9K-AS。转化P．pastorisGSll5,对获得的高拷贝转化子P．pastorisGSll5(pGAP9K-AS)进行组成型表达,同时以诱导型转化子P．pastoris GSll5(pPIC9K-AS)作为对照。SDS-PAGE结果显示：组成型转化子于培养4d后AS的表达水平已达到高峰,分泌量为58mg／L;而诱导型转化子诱导4d后表达的AS仅是组成型表达的70％,诱导6d后达到高峰,表达量也只是组成型表达系统表达高峰时(4d)的86％。CAM分析和抗癌实验结果显示：P．pastortis GS115(pGA．P9K-S)和P．pastoris GS115(pPIC9K-AS)表达的AS均具有抑制血管生成和C57BL／6J实验小鼠的B16黑色素瘤的生长,其平均瘤重抑制率分别达到90．61％和90．54％。以上结果表明,以GAP启动子构建的组成型表达系统具有发酵时间较短、表达水平较高、不用甲醇诱导、操作系统比较简单等优点,PGAP可以取代PAOX1在P．pastoris中表达AS及其他外源蛋白。