嗜酸乳杆菌质粒的分析和穿梭载体的构建

为分析嗜酸乳杆菌内源性质粒pDX，并基于该质粒构建大肠杆菌-嗜酸乳杆菌穿梭载体。本研究从嗜酸乳杆菌XW118中分离获得内源性的质粒pDX，对其进行序列测定及功能分析，然后利用质粒pDX的复制子构建了能在大肠杆菌和乳酸菌中穿梭的载体，并研究嗜酸乳杆菌的内源性质粒启动子在大肠杆菌中的活性和穿梭载体在乳酸菌中宿主的范围、转化效率和传代稳定性。结果显示嗜酸乳杆菌质粒pDX大小为2062 bp,GC含量为38.2%，包含4个开放阅读框(Open Reading Frame,ORF)，推定为滚环复制质粒。质粒pDX在嗜酸乳杆菌中的拷贝数最高为31.05。本研究构建了基于质粒pDX复制子和启动子的大肠杆菌-乳酸菌穿梭载体。该载体可转化多种类型的乳酸菌，转化效率在0.24×10²～2.75×10³ CFU/μg(质粒量)之间，质粒丢失率在25.45%～48.36%之间。本研究通过构建获得能在大肠杆菌-乳酸菌穿梭的载体，并获得了在大肠杆菌中具有活性的乳酸菌内源性质粒的启动子，为乳酸菌基因工程和大肠杆菌代谢工程提供了新的操作途径。     

植物乳杆菌内源性质粒序列分析及其表达载体的构建

目的:构建乳酸杆菌的表达载体,对菌株携带的质粒进行序列测定和功能分析,并利用自身带有的质粒系统构建乳酸杆菌的表达系统,对乳酸菌口服疫苗的制备起到积极的促进作用。方法:从植物乳杆菌LP3中提取质粒并进行序列测定和功能分析。通过质粒pLP3复制子,与pCPSP载体的氯霉素基因CmR、pUC19的复制子构建大肠杆菌-乳酸菌穿梭质粒。在穿梭质粒的基础上加上嗜酸乳杆菌的S层蛋白启动子PslpA和绿色荧光蛋白(green fluorescent protein,eGFP)基因,进一步构建乳酸菌表达载体D-pLP3-PslpA-eGFP。结果:从植物乳杆菌LP3中得到一个天然隐蔽性质粒pLP3,该质粒大小为2 017 bp,GC含量为37.48%。基于质粒pLP3,构建了大肠杆菌-乳酸菌穿梭质粒D-pLP3,并成功转化至不同类型乳酸菌,转化效率介于0.3×10~2～1.0×10~3 CFU/μg(DNA质量计)之间。乳酸菌表达载体D-pLP3-PslpA-eGFP转化至植物乳杆菌,成功获得了荧光蛋白的表达。结论:成功构建了乳酸菌表达载体D-pLP3-PslpA,为乳酸菌基因操作提供了新的工具。     

植酸酶YiAPPA在粪肠球菌中的高效组成型表达研究

旨在获得强组成型启动子来实现植酸酶YiAPPA在粪肠球菌EXW27中的高效组成型表达。本研究通过对比分析粪肠球菌中16S rRNA的启动子序列,将启动子中非保守区域替换为随机化序列,获得随机的人工启动子序列。然后利用大肠杆菌-乳酸菌穿梭质粒pSIP409中酶切位点构建人工启动子文库,并结合植酸酶活性的高通量筛选技术,筛选获得启动YiAPPA在粪肠球菌EXW27中高效组成型表达的强组成型启动子,实现YiAPPA在粪肠球菌EXW27中成功表达,并研究重组YiAPPA的酶学性质。结果表明,在组成型启动子p10的控制下,重组YiAPPA在粪肠球菌EXW27中成功表达,其表达量约占细胞内蛋白总量的15%。重组YiAPPA的最适反应pH为4.5,在pH1.0~10.0的范围内具有优良的pH稳定性,于55 ℃的绝对酶活高达3900 U/mg。本研究构建了既具有益生特性又具有植酸酶活性的转基因粪肠球菌,为研制新型转基因微生态制剂奠定了基础。     

组成型分泌表达蛋白酶的重组粪肠球菌的构建及应用

为构建组成型分泌表达蛋白酶Ker的重组粪肠球菌,采用豆粕固态发酵实验并评估其应用效果。以重组质粒pSIP401-kerhds为基础框架,向其中引入组成型启动子p10和高效分泌信号肽S6,构建重组载体pSIP401Z-s6-kerhds。采用电击转化方法将该重组载体转入具有益生特性的粪肠球菌EXW27来构建重组粪肠球菌。重组粪肠球菌的胞外蛋白酶的比酶活力为125.37 U/mL。重组蛋白酶Ker的最适反应温度为40℃,在pH 5.0~10.0范围内具有较高的相对酶活力和稳定性,并对胆盐溶液具有较好的耐受性,可适用于动物肠道环境。豆粕固态发酵实验表明,与粪肠球菌EXW27相比,重组粪肠球菌可以更有效降解豆粕中蛋白质。该研究构建了既具有益生特性又具有蛋白酶活性的重组粪肠球菌,为开发新型微生态制剂奠定了基础。     

乳酸乳球菌内源隐蔽性质粒序列分析及食品级克隆载体的构建

为构建乳酸菌食品级载体,对乳酸乳球菌内源隐蔽性质粒p KL001进行全序列测定、序列分析及PCR扩增,获得了该质粒的复制子。以Southern杂交对质粒p KL001复制模式进行分析,结果显示p KL001为θ型复制。将该复制子与镉抗性功能片段连接,构建了食品级克隆载体p KF168,其在受体菌株KLDS4.0319-5中可稳定存在。     

乳酸菌隐蔽性质粒pEV105复制子的鉴定

将Tnl541转座子的红霉素抗性rRNA甲基化酶基因克隆至pUCl9的EcoO109 I位点 ,构建成能够确定乳酸菌质粒复制子所在片段的复制检测载体pUB380.将乳酸菌隐蔽性质粒pEV105 3个酶切片断(4.5、3.5、3 kb)分别连接至此载体,并电转化至原宿主菌.试验结果显示,连接了3.5 kb片段的栽体能够使原宿主菌表达红霉素抗性的片段,即复制子所在片段.以Southern杂交对质粒pEV105复制模式进行分析.结果显示pEV105为O型复制.用400μg/mL吖啶橙对菌株KLDS6.0718处理50代后没有质粒丢失,说明pEVl05在宿主菌内十分稳定.该复制子在构建乳酸菌食品级克隆表达载体中可作为稳定的复制子使用.     

乳酸乳球菌镉抗性菌株的鉴定及质粒消除的研究

为构建乳酸菌食品级载体/受体系统.从分离自内蒙古传统乳制品103株乳源性乳酸菌中筛选出7株高浓度镉抗性菌株.特异性16SrDNA PCR扩增产物的序列测定结果表明.其中两株为乳酸乳球菌.PCR扩增镉抗性基因部分序列证实了这两株乳酸乳球菌含有镉抗性基因,为镉抗性阳性菌株.通过物理和化学方法消除乳酸乳球菌内源质粒,确定了镉抗性质粒,并获得一系列衍生菌株,包括无质粒菌株.为进一步研究各种质粒的功能及基因表达提供了理想的宿主,也为乳酸菌食品级载体/受体系统的构建奠定了基础.     

乳酸菌质粒研究

乳酸菌质粒编码许多重要的代谢特性,对优良菌株的育种和食品级载体的构建均具有重要意义.本试验研究发现德氏乳杆菌保加利亚亚种、乳酸乳球菌乳酸亚种、嗜酸乳杆菌存在23kb的质粒;28kb的质粒也存在于乳酸乳球菌乳酸亚种中.乳酸菌属于革兰氏阳性菌,菌体裂解不彻底;质粒拷贝数低,提取比较困难.     

乳酸乳球菌两组分食品级NICE系统载体的构建

乳酸菌食品级NICE系统是目前较理想的可控制蛋白质生产的食品级诱导表达系统。本实验构建的食品级表达载体pRNA48含α-aga食品级选择标记、θ型复制子和nisA启动子，与宿主菌L．hctis NZ9000共同构成乳酸乳球菌食品级NICE系统，可用于目的基因在乳酸乳球菌中高效诱导表达。     

植物乳杆菌melA基因的克隆及其作为食品级筛选标记的初步研究

以L. plantarum1.557的基因组DNA为模板,通过PCR技术成功克隆α-半乳糖苷酶基因(melA基因),与报道的melA基因序列同源性达到99%以上。再以大肠杆菌-乳酸菌穿梭表达载体pMG36e为基本骨架,将melA基因插入该载体,构建melA基因标记的穿梭表达载体pMG36e-melA。重组表达质粒pMG36e-melA经Sac I和Sph I双酶切和PCR鉴定与预期片段大小相符。结果表明已初步构建了以melA基因为食品级筛选标记的重组载体。     

Determination of nucleotide sequence related to the plasmid replication region in Enterococcus faecalis and its application to a new shuttle vector

The 5.1-kb plasmid pAMalpha1delta2, a derivative of the 9.6-kb plasmid pAMalpha1 which is harbored by Enterococcus faecalis ATCC 14508, has a region necessary for replication in E. faecalis. The nucleotide sequence related to the replication region in pAMalpha1delta2 was determined and found to contain an open reading frame of 720-bp encoding a replication protein. The sequence showed 54.5 and 48.5% homology to those encoding the RepAs of plasmids pLA103 from Lactobacillus acidophilus and pFA3 from Neisseria gonorrhoeae, respectively. A recombinant 5.8-kb plasmid, pEFX6, which can be used as a shuttle vector between Escherichia coli and some strains of E. faecalis, was constructed by combining the tetracycline resistance gene of pAMalpha1delta2 and the replication regions of pAMalpha1delta2 and pUC18 for E. faecalis and E. coli, respectively. This shuttle vector was successfully used to clone and express the gelatinase gene from E. faecalis subsp. zymogenes IFO 3989 in E. faecalis C57, a strain showing no gelatinase activity.     

组成型分泌表达嗜热酸性生淀粉α-淀粉酶的重组粪肠球菌的构建

该研究以高效组成型启动子P10替换pSIP401-gtamyhds中诱导型启动子,构建重组载体pSIP401Z-gtamyhds,并以其为基础框架,分别导入粪肠球菌（Enterococcus faecalis）来源的8种信号肽（s1～s8）,采用电转化法将信号肽筛选载体转入具有优良益生特性的粪肠球菌EXW27,构建组成型分泌表达嗜热酸性生淀粉α-淀粉酶Gt-amy的重组粪肠球菌。以发酵上清液中α-淀粉酶活性为评价指标,对8种信号肽进行筛选,并对嗜热酸性生淀粉α-淀粉酶Gt-amy进行分离纯化和酶学性质研究。结果表明,信号肽s7对嗜热酸性生淀粉α-淀粉酶Gt-amy的分泌效率最高,发酵上清液中α-淀粉酶活性达到310 U/mL。重组Gt-amy的最适反应pH值为5.0,在pH 4.0～8.0范围内具有较好的稳定性,最适反应温度为80 ℃,于80 ℃的半衰期为3 h,在40 ℃条件下反应3 h,对玉米淀粉的降解率可达55.8%。     

Construction of an autonomously replicating plasmid in n-alkane-assimilating yeast, Candida tropicalis

A transformation system using the autonomously replicating plasmid in the n-alkane-assimilating and asporogenic diploid yeast, Candida tropicalis, was developed. For the cloning of a DNA fragment containing a potential autonomously replicating sequence (ARS) from the genomic DNA of C. tropicalis, the ura3 mutant obtained using ethylmethane sulfonate as the host and the URA3 gene amplified by PCR using the C. tropicalis genomic DNA as a selectable marker were prepared. Comparison of ARSs among yeasts revealed that the consensus sequence found in S. cerevisiae was also present in C. tropicalis. The autonomously replicating plasmid containing the putative ARS as the shuttle vector, capable of replicating in both E. coli and C. tropicalis, was first constructed. The transformation system using this plasmid, in addition to the integrative transformation system, will be applicable to genetic studies of C. tropicalis.     

In vivo cloning by homologous recombination in yeast using a two-plasmid-based system

In order to reduce the number of classical DNA manipulation and ligation steps in the generation of yeast expression plasmids, a series of vectors is described which facilitate the assembly of such plasmids by the more efficient ‘recombination in vivo’ technique. Two sets of vectors were developed. The first set, called ‘expression vectors’, contains an expression cassette with a yeast promoter and the PGK terminator separated by a polylinker, and an Escherichia coli replicon. Subcloning in these vectors of a DNA fragment generates a ‘transfer vector’ which is compatible with the second set of E. coli–yeast shuttle vectors. This set of ‘recombination vectors’ contains a cassette for a functional copy of a gene complementing a host strain auxotrophy or a bacterial gene conferring an antibiotic resistance to the plasmid-bearing host. Plasmid copy numbers can be modulated through the use of URA3 or URA3-d as the selective marker together with an ARS/CEN and the 2 μm replicon. Integration of the cloned DNAs into the yeast linearized replicative vectors occurs by recombination between homologous flanking sequences during transformation in yeast or E. coli. All the vectors contain the origin of replication of phage f1 and allow the generation of single-stranded DNA in E. coli for sequencing or site-directed mutagenesis. The sequence presented (Figure 1a) has been entered in the EMBL data library under Accession Number Z48747.     

酵母细胞的高效转化方法

运用醋酸锂处理酵母细胞，建立并优化了酵母完整细胞的高效质粒转化体系，转化率达１０４μｇ－１．通过对影响转化的诸因子的研究，发现载运ＤＮＡ是影响酵母完整细胞转化效率的最主要因素，热休克处理及处理时间、聚乙二醇相对分子质量及浓度等对转化率也有显著影响。     

ISOLATION AND CHARACTERIZATION OF BACTERIOCIN-PRODUCING MICROORGANISMS FROM AGOS-OS

Agos-os, a fermented meat and sweetpotato mixture, was produced and analyzed for its microbial characteristics. pH decreased during fermentation. Mold and anaerobic bacterial counts increased while yeasts and aerobic bacterial counts decreased during the third and seventh day of fermentation. Six isolates with the widest zones of inhibition on the indicator lawn were selected for bacteriocin production. These isolates had exactly the same morphological, physiological and biochemical characteristics. The ribosomal RNA sequence was 99.5% identical with Enterococcus faecalis VRE 1492. The identification was confirmed through DNA homology test by the EMBL Genbank, Canada. This bacterium produced the L-isomer lactic acid. The amount of bacteriocin produced by the bacterium was optimized by growing the bacterium at different growth media, initial pH and fermentation time. Maximum production of bacteriocin was achieved in MRS (De Man Rugosa and Sharpe) medium (with glucose) at pH 7.50. The crude bacteriocin inhibited the growth of gram-positive bacteria such as Lactobacillus sake 15521 and Listeria innocua. The gram-negative bacteria such as Escherichia coli DH 5-alpha (with plasmid, PUC) , Salmonella typhii and Staphylococcus aureus were weakly inhibited. Other microorganisms such as Lactobacillus curvatus D31685 , Lactobacillus confusius M23036 , Lactococcus lactis MG1363 , Leuconostoc paramesenteroides S67831 , Pediococcus pentosaceus M58834 , Saccharomyces cerevisiae SS553 (wild type) and Escherichia coli JM109 (no plasmid) were not inhibited.     

Expression of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) by Hansenula polymorpha.

The methylotrophic yeast Hansenula polymorpha, a host organism for the production of heterologous proteins, has been applied to produce the alpha-galactosidase from the plant Cyamopsis tetragonoloba (guar). The yeast/Escherichia coli shuttle expression vector used is based on the origin of replication of the endogenous 2 microns plasmid of Saccharomyces cerevisiae and the LEU2 gene of S. cerevisiae for selection in H. polymorpha. In the expression vector, the alpha-galactosidase is controlled by the methanol-regulated promoter from the methanol oxidase gene, MOX, of H. polymorpha. The signal sequence of SUC2 (invertase) from the yeast S. cerevisiae, was used to ensure secretion of the alpha-galactosidase enzyme. After transformation and stabilization, the expression vector was stably integrated in the genome. The active alpha-galactosidase enzyme was efficiently secreted (greater than 85%) and after methanol induction, the expression level was 42 mg/l. Amino-terminal sequencing of the purified alpha-galactosidase enzyme synthesized by H. polymorpha showed that the S. cerevisiae invertase signal sequence was correctly processed by H. polymorpha. The secreted alpha-galactosidase was glycosylated and had a sugar content of 9.5%. The specific activity of the alpha-galactosidase produced by H. polymorpha was 38 U mg-1 compared to 100 U mg-1 for the guar alpha-galactosidase. Deglycosylation of the H. polymorpha alpha-galactosidase restored the specific activity completely.