无痕敲除法构建食品安全级枯草芽孢杆菌无芽孢菌株

利用同源重组法快速构建枯草芽孢杆菌(Bacilus subtilis168)spo0A基因缺陷型无芽孢菌株B.subtilis168Δspo0A。经PCR及核酸电泳验证,孢子形成率鉴定和芽孢染色镜检,确定最终获得不产芽孢的B.subtilis168Δspo0A基因缺失突变菌株。基因敲除完成后,菌株基因组不需引入抗性基因或其它任何筛选标记,实现了对spo0A基因的无痕基因敲除。本研究对枯草杆菌的工业化生产及提高相关产品食品质量安全性具有重要意义。     

群体感应系统对单增李斯特菌生物被膜形成的影响

以单增李斯特菌（Listeria monocytogenes）为研究对象,分析Agr群体感应系统和LuxS/AI-2系统对其生物被膜形成的调控作用。通过同源重组对LMB33426菌株进行agrD基因以及luxS基因的无痕敲除,比较野生菌株与agrD、luxS基因缺失菌株的生物被膜特性差异。结果表明,与野生株相比,ΔagrD突变株和ΔluxS突变株的生物被膜形成能力下降;ΔagrD突变株的疏水性显著下降,在37 ℃下的泳动能力较野生株增强;群体感应系统基因敲除对菌株的耐药性没有产生较大影响。基因缺失株的构建为进一步研究群体感应系统对单增李斯特菌生物被膜形成的调控机制提供参考,同时为单增李斯特菌的预防控制奠定了基础。     

一种谷氨酸棒杆菌基因无痕敲除载体的构建及应用

以谷氨酸棒杆菌（Corynebacteriumglutamicum）23604编码赖氨酸胞内转运蛋白基因lysP为敲除对象,利用重叠PCR技术将 lysP基因的上下游同源臂融合,并构建了由木糖启动子Pxyl和毒素蛋白基因mazF组成的筛选盒,同时无缝克隆连接至带有卡那霉素抗性基因的pTOPO载体中,经电转化及两次同源单交换筛选,实现lysP基因的无痕敲除。 结果表明,经过卡那霉素抗性筛选、木糖二次筛选及基因组PCR鉴定后,成功获得lysP基因缺失菌株C.glutamicum23604ΔlysP;发酵后C.glutamicum23604ΔlysP赖氨酸产量较对照菌株产量提高了10.8%。该实验以大肠杆菌毒素蛋白基因mazF作为反向筛选标记的敲除载体,实现谷氨酸棒杆菌lysP的高效敲除; lysP基因的敲除使得赖氨酸不能进入胞内而在胞外积累,从而达到增产赖氨酸的目的。     

米曲霉3.042尿苷/尿嘧啶营养缺陷型遗传转化体系的构建

米曲霉对潮霉素等多种抗生素不敏感,对其基因改造有一定困难。该研究以米曲霉3.042为出发菌株,建立pyrG筛选标记遗传转化体系。首先,分别运用紫外诱变法和左右臂同源重组法破坏米曲霉自身的pyrG基因,在含有5-氟乳清酸和尿苷/尿嘧啶的培养基平板进行筛选,最终两种方法分别获得性状稳定的尿苷/尿嘧啶营养缺陷型突变株米曲霉O11和米曲霉g-1。随后以缺陷型突变株为出发菌株,利用农杆菌转化法转入包含pyrG回补标记的质粒,其中米曲霉g-1突变株获得原养型的回补菌株,紫外诱变法获得的缺陷型突变菌株米曲霉O11未能恢复为原养型。研究表明米曲霉3.042 pyrG基因大片段缺失的缺陷型菌株,可直接以自身pyrG基因作为选择标记进行遗传改造。     

热带假丝酵母肉毒碱乙酰基转移酶基因的删除及功能鉴定

缺乏高效基因删除技术是限制热带假丝酵母菌株代谢工程育种的重要因素。论文发展了一种新型的基因删除技术并利用该技术成功删除了肉毒碱乙酰基转移酶的两个等位基因。首先PCR扩增标记基因(URA3)的3'端324 bp序列作为基因删除辅助序列(gda序列),同向插入到URA3基因的5'端,在此基础上构建两端含有CAT基因同源臂序列的基因删除突变盒,转化宿主菌XZX,获得CAT基因单拷贝和双拷贝敲除的突变株。PCR鉴定和DNA测序结果表明,获得的URA3基因弹出后的突变株,仅在基因组重组位点上残留一个gda序列。进一步鉴定了突变株的生理性能,单拷贝敲除菌株CAT活性较亲本下降80%,但在葡萄糖或烷烃上的生长性能并不受显著影响。双拷贝缺失菌株CAT活性完全丧失,不能利用烷烃生长,同时在葡萄糖培养基中的生长受到显著影响,最终生物量达到7.56(OD600),仅为出发菌株的57.8%。     

米曲霉RIB40高效同源重组和尿苷/尿嘧啶营养缺陷型菌株的构建

目的:以米曲霉RIB40为出发菌株，构建具有高同源重组效率的营养缺陷型菌株。方法:首先通过同源重组技术和5-氟乳清酸（5-Fluoroorotic Acid，5-FOA）对尿苷/尿嘧啶营养缺陷的筛选作用，获得AopyrG基因缺失的米曲霉RIB40。然后利用烟曲霉AfpyrG基因替换Aoku70得到?Aoku70敲除菌株，再通过5-FOA的筛选作用使得?Aoku70菌株基因组发生自身环化，丢失AfpyrG基因。最后为了检验?Aoku70?AopyrG菌株的可用性，将编码红色荧光蛋白的mCherry基因敲入至双突变菌株的组蛋白H2B基因上，并通过激光共聚焦显微镜观察红色荧光蛋白质的亚细胞定位。结果:通过AopyrG和Aoku70的双基因敲除，获得了高同源重组效率的营养缺陷型菌株?Aoku70?AopyrG。用激光共聚焦观察到红色荧光蛋白定位于细胞核，表明?Aoku70?AopyrG菌株可用于米曲霉的基因改造。结论:?Aoku70?AopyrG菌株可作为一个高同源重组效率的营养缺陷型出发菌株用于今后米曲霉RIB40的遗传改良。     

采用同源重组技术构建金黄色葡萄球菌sae基因缺失突变株

针对金黄色葡萄球菌sae基因前后两段序列设计两对引物,PCR扩增出sae基因上下游同源臂序列,克隆到穿梭载体pBT2中;两段序列之间用来自质粒p646的Em抗性基因片段连接,作为筛选标记,从而构建同源重组穿梭质粒pBT2△sae;将pBT2△sae电转化到金黄色葡萄球菌菌株RN4220中,40℃经过七轮培养,进行抗性培养基筛选和PCR验证,以及RT-PCR观察基因表达水平.获得一株金黄色葡萄球菌sae基因缺失突变株RN△sae,为进一步研究sae基因的调控机制等提供有用的实验材料.     

单核细胞性李斯特菌基因dal的敲除及其生物学特性初步分析

在单核细胞性李斯特菌(Listeria monocytogenes)野生株EGDe act A及inl B双基因缺失株(EGDeΔact AΔinl B)的基础上,利用同源重组的方法进一步构建了缺失营养基因dal的菌株(EGDe Δact AΔinl BΔdal),并对该缺失菌株生长状态、毒力基因表达水平、生物被膜的形成量及细胞侵袭等方面作进一步分析。结果显示,37℃摇床培养6 h后,缺失株的菌浓度显著低于EGDe Δact AΔinl B(P0.001),培养基中补充D-丙氨酸的缺失株生长速率与亲本株相比无显著差异;实时荧光定量聚合酶链式反应结果显示,缺失株的sig B基因表达水平变化最明显(P0.01),约下调90%;缺失株生物被膜形成量显著增加(P0.05),培养基补充D-丙氨酸后缺失株生物被膜的生成量与亲本株相比无差异;对Coca-2细胞的侵袭无影响,表明该基因对细菌生长能力及生物被膜形成具有重要的调控作用,并不影响菌株对细胞的侵袭力。此缺失株的构建为进一步研究基因dal的功能提供了理论支持。     

多倍体啤酒酵母URA3基因的失活与恢复

以多倍体啤酒酵母菌株S6作为出发菌株,利用同源重组的方法构建了尿嘧啶营养缺陷型啤酒酵母菌株S6-ΔU,使之用于遗传标记,并把缺陷型菌株恢复成原营养型S6'。同时对出发菌株S6,缺陷型菌株S6-ΔU和恢复后的菌株S6'的生长状况进行比较。结果表明,URA3点突变并没有弱化菌株的生长状况。从而建立一种快速获得缺陷型啤酒酵母菌株的方法。     

BAT2缺失酿酒酵母基因工程安全菌株的构建及其杂交育种

为了获得低产高级醇基因工程安全菌株,首先将带有Cre重组酶编码基因的pGAPZA质粒转入BAT2缺失单倍体突变株A8-B和C22-B中,利用Cre/loxp系统去除G418抗性基因(KanMX),获得酿酒酵母单倍体突变株ΔBAT2a和ΔBAT2α。然后将这2株单倍体杂交成双倍体菌株ΔBAT2,该菌株具有良好的遗传稳定性。以野生菌株AY15和2株单倍体出发菌株为对照,对基因工程安全菌株进行白酒发酵试验,实验结果显示,基因工程安全菌株ΔBAT2与出发菌株相比,CO2失重高,残糖低,乙醇含量高;与野生菌株AY15相比,异丁醇、异戊醇、总高级醇生成量分别降低50.00%、33.40%和30.57%。     

Studies of yeast Kluyveromyces lactis mutations conferring super-secretion of recombinant proteins

We have isolated mutants responsible for a super-secretion phenotype in Kluyveromyces lactis using the gene coding for a Bacillus amyloliquefaciens alpha-amylase as a marker for secretion. These mutations defined two groups, dominant and recessive. The recessive mutant strain, which secreted the heterologous protein in five-fold excess compared to the wild-type strain, was used for the cloning of genes, restraining the super-secreting phenotype. In screening for genes affecting super-secreting phenotype, we found that multiple copies of 10 different independently isolated DNA sequences suppressed the super-secreting phenotype. The first among the genes characterized, named KlSEL1 ('secretion lowering') showed homology to Saccharomyces cerevisiae ORF YML013w. The KlSEL1 gene is predicted to encode a polypeptide of 620 amino acid residues containing a putative transmembrane domain and UBX domain, characteristic for the ubiquitin-regulatory proteins. We demonstrated that the disruption of the SEL1 orthologues in K. lactis and S. cerevisiae conferred the super-secreting phenotype. SEL1 isolated from S. cerevisiae suppressed the super-secretion phenotype in K. lactis klsel1 strain, likewise homologous KlSEL1. No other phenotypic features for strains lacking the SEL1 gene were noticed except for the S. cerevisiae mutant growth being notably slower than in a wt strain. No growth changes were observed in the K. lactis klsel1 mutant. The set of genes (suppressors of over-secreting phenotype) could be attractive for further analysis of gene functions, super-secreting mechanisms and construction of new strains. This collection could be useful for the expedient construction of reduced yeast genomes, optimized for heterologous protein secretion.     

Expression and secretion of a prokaryotic protein streptokinase without glycosylation and degradation in Schizosaccharomyces pombe

Streptokinase (SK) is an important thrombolytic protein that is secreted by pathogenic strains of Streptococcus. Expression of streptokinase has been so far attempted in Pichia pastoris, Escherichia coli and Bacillus subtilis and shown to yield protein that was either highly glycosylated or degraded. Since the fission yeast, Schizosaccharomyces pombe, shares several molecular characteristics with higher eukaryotes, we decided to express the streptokinase gene in this yeast. A chimeric gene comprising the signal sequence of the Plus pheromone of Sz. pombe fused in-frame with the mature streptokinase from Streptococcus sp. was constructed and inserted into the expression vector containing the thiamine-regulated promoter. We obtained a high level of expression of streptokinase comparable to that in E. coli and P. pastoris, with 50-100% processing of the signal sequence and secretion of the mature streptokinase into the periplasmic fraction. The mature enzyme co-migrates with the authentic mature SK in SDS gels, lacks any major modification and is functional. Importantly, a higher level of expression under stationary phase conditions and improved extractability of the mature and undegraded streptokinase was achieved in a novel mutant of Sz. pombe defective for a potent extracellular protease activity. We suggest that the unique vector/strain system developed here could be advantageous for large-scale production of prokaryotic proteins without significant modification or degradation in Sz. pombe.     

DNA transformations of Candida tropicalis with replicating and integrative vectors.

The alkane-assimilating yeast Candida tropicalis was used as a host for DNA transformations. A stable ade2 mutant (Ha900) obtained by UV-mutagenesis was used as a recipient for different vectors carrying selectable markers. A first vector, pMK16, that was developed for the transformation of C. albicans and carries an ADE2 gene marker and a Candida autonomously replicating sequence (CARS) element promoting autonomous replication, was compatible for transforming Ha900. Two transformant types were observed: (i) pink transformants which easily lose pMK16 under non-selective growth conditions; (ii) white transformants, in which the same plasmid exhibited a higher mitotic stability. In both cases pMK16 could be rescued from these cells in Escherichia coli. A second vector, pADE2, containing the isolated C. tropicalis ADE2, gene, was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of the ade2 locus. Different integration types were observed at one or both ade2 alleles in single or in tandem repeats.     

米曲霉pyrG 基因克隆及其同源转化系统的建立

建立以乳清酸核苷-5＇- 磷酸脱羧酶基因(pyrG)为选择标志,以米曲霉为宿主菌的同源转化系统。以米曲霉基因组为模板,通过PCR 扩增获得pyrG 基因,将该片段与表达载体pMD18-T 相连,转化大肠杆菌DH 5α,经蓝白斑筛选、PCR 快速筛选、酶切和测序验证,获得重组质粒pMD-pyrG,完成pyrG 基因的克隆。序列分析表明该基因与米曲霉KBN616 的pyrG 基因编码序列的同源性为99.9%,两者经推导的氨基酸的同源性为99.6%。以米曲霉 pyrG 营养缺陷株为受体菌,通过PEG/CaCl2 诱导的原生质体转化方法,将重组质粒导入该受体菌,使米曲霉pyrG 缺陷株发生基因转化,成为pyrG+,由此成功建立了以pyrG 为筛选标记基因、同型米曲霉pyrG 基因缺陷株为受体菌的基因转化系统。     

Genetic labeling of lactobacilli in a food grade manner for strain-specific detection of industrial starters and probiotic strains

In order to demonstrate that creation of silent mutations allows a precise strain-specific detection with polymerase chain reaction, silent mutations positions were introduced in adjacent codons of the Lactobacillus helveticus CNRZ32 chromosomal pepX gene without altering the amino acid sequence of the gene product, X-prolyl dipeptidyl aminopeptidase (PepX). The mutated pepX gene was constructed with polymerase chain reaction and cloned with the thermosensitive pSA3 plasmid in L. helveticus CNRZ32 and integrated in the chromosomal DNA by homologous recombination at a restrictive temperature. Gene replacement of the wild-type pepX gene with that of the mutated variant was obtained in the second homologous recombination event. The PepX activity of mutant strains carrying the pepX gene with silent mutations did not differ from that of the wild-type strain. PCR was performed with strain-specific primers from milk and faecal samples spiked with the wild-type or mutant L. helveticus strain. For this purpose, a method for removing PCR inhibitors from faecal samples by washing steps and cell mill disruption of the bacterial cells combined with Sepharose CL-6B spin column chromatography was also developed.     

基于Red重组系统的阴沟肠杆菌基因重组技术

目的:开发一种基于Red重组系统的E.cloacae基因重组技术。方法:将Red重组酶基因连接于表达载体形成pSC-MSC-red,将Flp重组酶基因连接于表达载体形成pSC-MSC-flp。以budA基因(编码乙酰乳酸脱羧酶)为例,构建了不同长度同源臂的抗性盒。将这些DNA片段分别转化入携带pSC-MSC-red质粒的E.cloacae进行基因重组。结果:使用同源臂长度为39和100 bp的抗性盒不能得到重组子;同源臂长度为200 bp的抗性盒可以获得重组子E.cloacae ΔbudA-773,重组效率为6.1 CFU/μg DNA;同源臂长度为500 bp时,重组效率提高到131.5 CFU/μg DNA。将表达Flp重组酶的质粒pSC-MSC-flp转化入重组菌株中传代培养成功消除了抗性标记。对重组菌株E.cloacae ΔbudA进行发酵培养实验,菌株丧失了合成乙偶姻和2,3-丁二醇的能力,表明budA基因被成功敲除。结论:本文建立了一种适用于E.cloacae的基因重组方法。     

单核增生性李斯特菌hly缺失菌株的构建及生物特性的初步鉴定

为研究单核增生性李斯特菌(Listeria monocytogenes,Lm)hly基因对毒力的影响,利用同源重组原理,使用穿梭载体,构建单核细胞增生性李斯特菌野生菌株EGD-e的hly基因缺失菌株EGD-eΔhly。细菌活性实验证明缺失菌株生长状态与亲本无差异,但EGD-eΔhly丧失溶血活性,细胞侵袭能力降低约90%,在10~7 cells/m L腹腔注射条件下不表现动物毒性。在转录水平上EGD-eΔhly的毒力基因inl C、prf A的表达量分别下降了81%和76%,act A、plc B的表达量分别提高了2.7倍和1.8倍。hly缺失菌株的成功构建及生物特性的初步研究结果为研究Lm致病机理提供依据。     

里氏木霉RL-P37尿嘧啶营养缺陷型菌株的构建

里氏木霉（Trichoderma reesei） RL-P37作为纤维素酶高产菌,被广泛地应用在工业蛋白的生产过程.从基因水平对工业菌株进行分子改造已经成为提升工业菌株生产性状的重要手段.因此,亟需构建适用于基因敲除以及外源蛋白表达的营养缺陷型菌株,特别是可用于基因无痕敲除的尿嘧啶营养缺陷型菌株.本研究以潮霉素为标记,成功构建了里氏木霉RL-P37尿嘧啶营养缺陷型菌株,并得到Southern验证.该菌株在含有1.5 mg/mL 5-氟乳清酸、2 mg/mL尿苷的MM培养基中可以正常生长,并且在不含有尿苷的MM培养基上不能生长.此外,利用粗糙脉孢菌（Neurospora crassa）的pyr4基因对该突变体进行了回补.里氏木霉RL-P37尿嘧啶营养缺陷型菌株的构建为该菌纤维素酶表达和分泌的机理研究以及后续产酶性能分子改造奠定了物质基础.