葡萄糖异构酶突变体酶GIG138P和GIG138P-G247D在变铅青链霉菌中的高效表达及检测

将含有G138P单点突变和G138P-G247D双点突变的GI结构基因,分别克隆入E.coli链霉菌穿梭载体pHZ1272,成功构建了穿梭表达载体pHZGI1和pHZGI2。通过原生质体的转化,将穿梭表达载体导入变铅青链霉菌TK54菌株。30℃振荡培养24h,加入2 μg/mL 硫链丝菌素诱导表达12h。SDSPAGE电泳表明,两个穿梭载体在TK54菌株内表达出425 kD特异性条带。薄层扫描显示,突变体酶GIG138P和GIG138PG247D分别约占可溶性蛋白的19%和22%。Western杂交进一步证实GIG138P和GIG138PG247D在变铅青链霉菌TK54中获得了表达。     

单点突变葡萄糖异构酶(GIG138P)基因在变铅青链霉菌中的高效表达及其稳定性研究

通过三步亚克隆 ,将单点突变葡萄糖异构酶 ( GIG1 38P)基因及其调控序列插入链霉菌质粒p IJ40 83,构建重组表达质粒 p IJ40 83- GI1 .用重组质粒转化变铅青链霉菌 TK54原生质体 ,经硫链丝菌素抗性 ( Th R)筛选 ,获得重组菌株 TK54/p IJ40 83- GI1 .酶活力测定和 SDS- PAGE分析表明 ,GIG1 38P基因在变铅青链霉菌中得到高效表达 ,GI1粗酶液比活力为 1 5U/mg,GI1表达量约占菌体可溶性蛋白的 2 5% .同时也研究了重组质粒的遗传稳定性 .重组菌株在无选择压力条件下经液体连续传代培养 ,GI1比活力和 GI1表达量在 2 0 0 h传代时间中呈平缓下降趋势     

天蓝色链霉菌孢子形成的关键基因——whiG的诱导表达

与链霉菌分化有关的whiG结构基因已亚克隆到链霉菌表达载体pAK203的tipA启动子下游.该启动子能被硫链丝菌素诱导.whiG基因的诱导表达不仅可使天蓝色链霉菌孢子形成缺陷突变株C71恢复产生孢子的能力,而且也能使天蓝色链霉菌野生型菌株J1501和变铅青链霉菌TK54的孢子形成更为丰满.Western杂交也进一步证实了诱导表达后whiG基因产物——σ~(whiG)产量的增加.这为依赖于σ~(whiG)RNA多聚酶的发育调控启动子体外转录的研究提供了有利条件.     

生米卡链霉菌变株丙酰化酶基因的克隆和表达

麦迪霉素产生菌生米卡链霉菌(streptomyces mycarofaciens)变株具有丙酰化酶活性,可以将螺旋霉素转化为丙酰螺旋霉素。为了进行丙酰化酶基因克隆,本实验以质粒pIJ702为载体通过鸟枪克隆法将变株DNA片段克隆至变铅青链霉菌TK54(Streptomyces livdansTK54),经薄层层析和高压液相色谱分析结果表明,在转化子中,N0．9菌株可以将螺旋霉素转化为丙酰螺旋霉素,这证明丙酰化酶基因已在变铅青链霉菌TK54中克隆并得到初步表达,№．9重组质粒插入DNA片段为4．16kb,经southern杂交表明确实来源于变株。此外还构建了N0．9重组质粒的限制酶酶切图谱。     

刺糖多孢菌中铵载体蛋白基因amtS的克隆及功能研究

目的:铵离子是细胞内合成各种核酸、氨基酸和辅助因子等含氮化合物的重要原料之一。微生物细胞膜上的铵载体蛋白介导了铵离子的转运。通过异源表达刺糖多孢菌中铵载体蛋白基因,研究其对链霉菌产孢能力和次级代谢产物产量的影响。方法:从刺糖多孢菌S04-41菌株中克隆铵载体蛋白基因amt S,通过接合转移导入天蓝色链霉菌M145和变铅青链霉菌TK24中,分析比较amt S基因的异源表达对其产孢能力和次级代谢产物产量的影响。结果:天蓝色链霉菌重组菌株M145/p MF-amt S和变铅青链霉菌重组菌株TK24/p MF-amt S中放线紫红素的产量分别提高了2.85倍和30.02倍。结论:刺糖多孢菌中的铵载体蛋白能够提高链霉菌中次生代谢产物的产量,为进一步研究该基因的功能与对刺糖多孢菌中多杀菌素合成的作用奠定了重要基础。     

大肠杆菌葡萄糖异构酶基因在变铅青链霉菌中的克隆与表达

本文用穿梭质粒载体pSE-3,进行了大肠杆菌葡萄糖异构酶基因在变铅青链霉菌中的克隆与表达。把含有1.6kb葡萄糖异构酶基因的pX1200(4.3kb)质粒与pGEM-3(2.9kb)质粒分别用EcoRI酶解,T4DNA连接酶连接,转化E.Coli HB101(Xy1~-,Neo(?)),得到的重组质粒被命名为pX1203(7.2kb);将pX1203与穿梭质粒载体pSE-3分别用HindⅢ酶解,T_4DNA连接酶连接,转化E.Coli HB101,在含有新霉素的木糖培养基上筛选到转化重组体,其重组质粒被命名为pSE×100(10.6kb);把pSE×100转化变铅青链霉菌TK54的原生质体,在硫链丝菌肽(50μg/ml)和新霉素(50μg/ml)的平皿上得到了重组体。经质粒提取,酶切分析,再转化和葡萄糖异构酶活性测定,结果表明,大肠杆菌的葡萄糖异构酶基因确实已在链霉菌中克隆和表达。     

变铅青链霉菌与放线菌噬菌体φHAU3相互关系的研究

变铅青链霉菌ＺＸ１,是由变铅青链霉菌ＪＴ４６经ＮＴＧ诱变后产生的一株修饰基因突变株,与其亲本ＪＴ４６相比,ＺＸ１除了与ＤＮＡ降解有关的基因发生了突变（Ｄｎｄ－,即该突变株的ＤＮＡ在含有Ｆｅ２＋的缓冲液中电泳不受到降解,而野生型变铅青链霉菌在同样条件下则遭到降解）以外,对噬菌体　ＨＡＵ３的抗性也随之消失,这项特征主要表现在噬菌斑大小和成斑单位（效价）上的显著变化。研究结果表明,噬菌体　ＨＡＵ３以同等的频率吸附野生型变铅青链霉菌及其突变菌株ＺＸ１,从　ＨＡＵ３基因组中没有克隆到被变铅青链霉菌识别的特异性靶位点;噬菌体ＨＡＵ３的ＤＮＡ也可以转染野生型变铅青链霉菌原生质,但其释放的噬菌体粒子只能感染突变菌株ＺＸ１,而不能感染野生型变铅青链霉菌。噬菌体ＨＡＵ３在突变株ＺＸ１中的繁殖遵循一步生长曲线,单菌释放量大约为１００,而　ＨＡＵ３感染野生型变铅青链霉菌后,则检测不到噬菌体的释放。     

鲑鱼降钙素在链霉菌中的分泌表达

利用聚合酶链式反应(PCR)技术分别扩增鲑鱼降钙素的编码序列(sCT-Gly)和抗生链霉菌melCl的信号肽编码序列及其上游的调控序列,将鲑鱼降钙素编码序列融合在melCl信号肽编码序列后,定向克隆到链霉菌质粒载体pIJ680中的新霉素磷酸转移酶基因的启动子(Paph)下游,得到的表达质粒pMS680转化变铅青链霉菌(Streptomyces lividans TK54)进行分泌表达。酶联免疫测定法(EIA)显示重组菌株在YEME培养基中的分泌表达水平于96h达到最高,表达量大于100μ/L培养液。实验表明表达产物能降低大鼠血清钙的浓度。高压液相色谱(HPLC)结果表明,表达产物中主峰的保留时间与鲑鱼降钙素标准品的保留时间基本一致。以上结果提示,鲑鱼降钙素在链霉菌中获得了成功的分泌表达。     

吸水链霉菌NND-52-C基因工程宿主载体系统的构建

吸水链霉菌NND-52-C菌株是大环内酯类抗生素-阿扎霉素B的高产菌株。采用原生质体转化技术,将来自变铅青链霉菌TK24菌株的pU702质粒转化吸水链霉菌NND-52-C菌株的原生质体,建立了吸水链霉菌NND-52-C菌株的基因工程宿主载体系统。确定了NND-52-C菌株原生质体制备和再生的条件,其原生质体形成率达到10^8／mL,再生率约为0．2％,转化率为10^2-10^3个转化子／μg质粒DNA。     

毒性T淋巴细胞相关抗原-4(CTLA-4)在链霉菌中的表达研究

应用两种链霉菌新型信号肽--vsi和gpp在常用工程菌变铅青链霉菌（Streptomyces lividans）中进行了CTLA-4的分泌表达研究,vsi信号肽与CTLA-4的融合片段克隆至链霉素-大肠杆菌穿梭质粒pUWL-219,同时gpp信号肽与CTLA-4片段在质粒pLNSP中融合,分别转化S.lividans TK24,获得重组菌株S.lividans[pUWL219-VC]和S.lividans[pLNSP/CTLA-4]。重组菌株的发酵上清液经SDS-PAGE及Western blotting分析结果表明：应用不同信号肽构建的两株工程菌均能表达分子量为13000重组蛋白,具有免疫活性。     

Site-Specific Integration of the Double-Mutation Glucose Isomerase (GIG138PG247D) Gene in Streptomyces lividans and Its Stable Expression

A recombinant expression plasmid pYH12, containing the double-mutation glucose isomerase (GIG138PG247D, GI2) coding gene and its natural regulatory sequence, was constructed for site-specific integration in Streptomyces. The resulting plasmid was introduced into Streptomyces lividans TK54 by protoplast transformation and two apramycin-resistance (Am^R) transformants, designated GY2 and BY7, respectively, were obtained further based on enzyme assays. These results for polymerase chain reaction (PCR), Dot blot, and recovery of cloned fragments from the transformant chromosome indicated that the GI2 gene was integrated into the S. lividans chromosome by site-specific recombination, and which was further verified by Southern blot. We found that the free form of plasmid pYH12 co-existing with the integrated form was present in S. lividans. SDS-PAGE analysis showed that the GI2 gene was expressed in S. lividans. The intracellular GI2 specific activity was 1.15 U/mg. The stability of integrants demonstrated that the cloned GI2 gene was stably integrated and expressed even in the absence of selective pressure. Received: 28 March 2001 / Accepted: 14 May 2001     

Heterologous Expression of the Single-Mutation Glucose Isomerase (GIG138P) Gene in Streptomyces lividans and Its Genetic Instability

A 1.3-kb PstI-BamHI fragment containing the single-mutation glucose isomerase (GIG138P, GI1) gene and its natural promoter was inserted into PstI-BglII linearized Streptomyces vector pIJ702. The ligation mixture was then introduced into Streptomyces lividans TK54 protoplasts; transformants were identified based on their thiostrepton resistance (Th^R) and insertional inactivation of the melanin phenotype; and three white colonies, XY-2, 6, and 9, harboring recombinant expression plasmid pYH703, were obtained. Enzyme assay and SDS-PAGE analysis indicated that the GI1 gene was expressed, the intracellular GI1 specific activity was 6 U/mg, and GI1 accounted for 20% of the soluble proteins in S. lividans. Restriction analysis and Southern blot of pYH703 showed the existence of plasmid deletion, presumably owing to the interaction between the mel and GI1 sequences. Continuous liquid cultures of the recombinant strain demonstrated that the GI1 specific activity and GI1 expression in S. lividans decreased, and more obviously under non-selective conditions. Received: 10 August 2000 / Accepted: 5 September 2000     

Cloning and expression of a nitroaryl reductase gene from Streptomyces aminophilus strain MCMB411 in E. coli JM109 and Streptomyces lividans TK64

A partial genomic library was prepared in E. coli JM109 using pBR322 as vector and 2.4 kb Sau 3A I chromosomal fragment, encoding a nitroaryl reductase (nbr A) gene, from Streptomyces aminophilus strain MCMB 411. From the library, 2.4 kb fragment was recloned in E. coli JM109 and S. lividans TK64 using pUC18 and pIJ702 as vectors respectively. The recombinant plasmids pSD103 and pSD105 expressed the reductase gene and exported the enzyme in periplasmic space of E. coli and in cytoplasm of S. lividans TK64. The proteins expressed by E. coli and S. lividans had the same molecular mass (70 kD) as that expressed by parent strain, which suggested that the enzyme was processed similarly by all strains. Activities of the enzymes cloned in E. coli JM109 and S. lividans TK64 containing recombinant plasmids pSD103 and pSD105 respectively were optimum at 30 degrees C and pH 9 and requirement of cofactors was same as that of the parent strain.     

Cloning of a DNA fragment from Streptomyces griseus which directs streptomycin phosphotransferase activity

DNA from Streptomyces griseus ATCC 12475 was partially digested with Sau3A and fragments were ligated into BglII-cleaved pIJ702. When the ligation mixture was used to transform protoplasts of Streptomyces lividans TK54, two transformants resistant to both thiostrepton and streptomycin were isolated. The hybrid plasmids pBV3 and pBV4 which they contained, carrying inserts of sizes 4.45 and 11.55 kbp respectively, each retransformed S. lividans to streptomycin resistance at high efficiency. Both plasmids hybridized to restriction digests of S. griseus chromosomal DNA in Southern blot experiments. In vitro deletion and sub-cloning experiments showed the sequence conferring streptomycin resistance to lie within a segment of 1.95 kbp. Extracts of TK54(pBV3) and TK54(pBV4) contained a streptomycin phosphotransferase similar to that in extracts of S. griseus. Streptomycin phosphotransferase activity appeared in extracts of S. griseus, TK54(pBV3) and TK54(pBV4) within 2 d of inoculation. When pBV3 and pBV4 were retransformed into S. griseus with selection for thiostrepton resistance, plasmid DNA of sizes corresponding to the incoming plasmids was found in the transformants. In these transformants the phosphotransferase appeared at 1.5 rather than 2 d, and reached a level over twice that of the original S. griseus strain.     

Increasing the thermostability of D-xylose isomerase by introduction of a proline into the turn of a random coil.

Thermostability can be increased by introducing prolines at suitable sites in target proteins. Two single (G138P, G247D) mutants and one double (G138P/G247D) mutant of xylose isomerase from Streptomyces diastaticus No.7, strain M1033 have been constructed by site-directed mutagenesis. With respect to the wild-type enzyme, G138P showed about a 100% increase in thermostability, and G247D showed an increased catalytic activity. Significantly, the double mutant, G138P/G247D displayed even higher activity than G247D and better heat stability than G138P. Its half life was about 2.5-fold greater than the wild-type enzyme, using xylose as a substrate. Molecular modelling suggested that the introduction of a proline residue in the turn of a random coil may cause the surrounding conformation to be tightened by reducing the backbone flexibility. The change in thermostability can, therefore, be explained based on changes in the molecular rigidity. Furthermore, the improvements in the properties of the double mutant indicated that the advantages of two single mutants can be combined effectively.     

Q20L及G247D定点突变对葡萄糖异构酶酶活和最适pH的改善

用双引物法对GI基因进行体外定点突变,构建了突变体Q20L和G247D。含突变基因的重组表达质粒pTKD-GIQ20L及pTKDGIG247D在E.coli K38菌株中表达。纯化的突变酶与野生型酶相比：(1) GIQ20L的最适反应温度下降5℃,热稳定性为野生型酶的78%,对底物的亲和性增强;(2) GIG247D的酶活提高约33%,最适pH下降0.6个单位,但热稳定性降低。初步分析认为,Gln 20位于α0～α1螺旋之间,其亲水侧链被Leu的疏水侧链取代后,分子表面增强的疏水作用,反而不利于蛋白质的稳定,使GIQ20L的热稳定性降低。Gly247是酶活性中心β折叠(242～247aa)的最后一个残基。引入电负性极强的Asp后,可能改变分子的静电场分布,影响了活性部位的电荷传递过程,使GIG247D酶活提高。引入的电荷,可能改变活性中心可解离基团的pKa,使其最适pH下降。另外Asp247的侧链在周围空间结构中显得过于拥挤,易与其他侧链产生排斥,由此影响到β-折叠的稳定性,接近亚基结合面的Asp247,可能进一步影响到亚基间相互作用的稳定性,最终导致酶热稳定性的降低。GI酶活和最适pH的改善更利于工业生产。     

Cloning and heterologous expression of the entire set of structural genes for nikkomycin synthesis from Streptomyces tendae Tü901 in Streptomyces lividans.

A genomic library from Streptomyces tendae raised in shuttle cosmid vector pKC505 was screened with a previously isolated 8-kb DNA fragment containing the orfP1 gene, which is involved in nikkomycin biosynthesis. The entire set of structural genes for nikkomycin synthesis was heterologously expressed in S. lividans TK23 by introducing recombinant cosmids p24/32 and p9/43-2, carrying inserts of about 31 and 27 kb, respectively, overlapping by 15 kb. S. lividans transformants synthesized nikkomycins X, Z, I, and J, which were identified by high-pressure liquid chromatography analyses of culture filtrates.     

Influence of disruption of the recA gene on genetic instability and genome rearrangement in Streptomyces lividans.

Streptomyces lividans TK23 gives rise to chloramphenicol-sensitive (Cml(s)) mutants at a frequency of about 0.5%. This is due to the frequent occurrence of very large chromosomal deletions removing the corresponding chloramphenicol resistance gene. A mutant in which the recA gene has been disrupted (S. lividans FrecD3 [G. Muth, D. Frese, A. Kleber, and W. Wohlleben, personal communication]) segregated about 70 times more chloramphenicol-sensitive mutants than the parental strain. An enhancement of the deletion frequency was responsible for this mutator phenotype. The amplifiable locus AUD1 has a duplicated structure in some S. lividans strains and is frequently highly amplified in some mutants generated by genetic instability. The chromosomal AUD1 is not amplified in strain TK23 because of the lack of one duplication. Nevertheless, AUD1-derived amplifiable units presenting the typical duplicated organization amplified very well in TK23 when carried on a plasmid. No amplification of these units was observed in the recA mutant. The ability to amplify was restored when the wild-type recA gene was introduced into the plasmid carrying the amplifiable unit. These results suggest that the RecA protein plays a role in reducing the level of genetic instability and chromosomal deletions and show that the recA gene is necessary to achieve high-copy-number amplification of AUD1.