HNO_2诱变选育链霉菌702菌株

以链霉菌702-20为出发菌株,经HNO2诱变处理,获得高产突变株。实验结果表明:HNO2处理20 m in对菌株的致死率可达83.10%,突变率高达14.13%,经过摇瓶筛选获得高产突变株20-29-148,产链霉素能力达到1.404 mg/mL,比出发菌株提高了37.65%。经传代培养考察,该突变菌株具有良好的遗传稳定性。     

产耐高温谷氨酰胺转胺酶菌株的快速筛选方法

目的:采用亚硝基胍(NTG)诱变结合96孔板高通量筛选方法筛选产耐高温谷氨酰胺转胺酶(MTG)的茂原链霉菌(Streptomyces mobaraensis)。方法:通过优化96孔板高通量测定MTG活性的方法、确定筛选温度和时间,建立了产耐高温MTG菌株的快速筛选方法;通过优化NTG诱变条件建立了筛选突变库;通过96孔板高通量初筛、摇瓶复筛获得了产耐高温MTG的突变株12-82,并通过摇瓶发酵对12-82所产MTG进行热稳定性分析。结果:采用2mg/ml NTG、p H8.0、60min的诱变条件获得突变株,将突变株的发酵上清液于70℃水浴7.5min,再在37℃空气浴、反应10min的条件下测定MTG活性,从5 200株突变株中筛选出5株产耐高温MTG的突变株,其中突变株12-82在50℃水浴60min以及70℃水浴1.5min的酶活残留率均比出发株高出近20%,且80℃保温2min仍有11.9%的酶活残留率。结论:利用NTG诱变结合96孔板高通量筛选的方法筛选到5株所产MTG热稳定性相对较高的突变株,其中突变株12-82在50℃、70℃和80℃的酶活残留率均有10%~20%的提高。这为高温食品加工领域所需耐高温MTG生产菌株的高效筛选提供了可行性方案。     

基因组重排与传统育种相结合选育大观霉素高产菌株

以壮观链霉菌(Streptomyces spectabilis)为研究对象,采用基因组重排技术与传统诱变育种相结合的方法选育大观霉素的高产菌株.通过原生质体紫外诱变获得壮观链霉菌突变体群体,高产突变菌株间进行两轮的基因组重排,筛选的高产菌株用NTG诱变得新霉素和链霉素的抗性突变菌株,抗性突变菌株间进行两轮基因组重排,从...     

梅岭霉素产生菌抗药性突变标志诱变筛选模型的初步研究

本文通过梅岭霉素 (Meilingmycin)产生菌南昌链霉菌NS 4 1 80菌株孢子对 6种抗生素敏感性测定 ,采用诱变剂EMS四种不同诱变剂量对菌株孢子进行诱变处理 ,诱变处理的孢子涂布在含致死浓度链霉素的高氏平板上。然后从抗药性突变标志菌株中进一步筛选梅岭霉素高产菌株。在 150 0多个抗药性突变株中通过初筛获得了比诱变出发菌株的产素能力提高 50 %以上菌株。通过诱变剂量分别与抗药性突变率和突变株产素产量的变势统计分析表明 ,菌株抗药性突变与产素突变密切相关 ,产素突变的EMS诱变剂量高于抗药性突变诱变剂量 ,在 0 .0 3mol/LEMS剂量作用下 ,菌株致死率为 99.4 3% ,抗药性突变率为 0 .0 4 4 0 % ,建立了梅岭霉素产生菌抗药性突变标志诱变推理性筛选模型。为南昌链霉菌高产菌种选育研究作了有益的尝试 ,并有助于其它链霉菌属的抗生素产生菌育种研究。     

复合诱变选育林肯霉素高产菌株

以林肯链霉菌9502(Streptomyces lincolnensis9502)为出发菌株,进行NTG诱变处理,并用高效的琼脂块培养法对菌株进行筛选,得到产林肯霉素相对效价提高35.4%的变异株9502-7。对9502-7菌株孢子采用紫外线处理,得到变异高产菌株9502-7-12,其相对效价较出发菌株提高50%以上。     

磷脂酶D高产菌株的原生质体紫外诱变选育

为了获得磷脂酶D高产菌株,由链霉菌野生菌株LD0501出发研究原生质体的制备和再生条件,建立原生质体紫外诱变筛选方案。采用酶解法制备原生质体,用紫外线对原生质体诱变,TLC检测突变株产磷脂酶D活力。原生质体的适宜条件:种子培养基中甘氨酸质量浓度5 g/L,菌龄72 h,用3 mg/m L的溶菌酶在30℃下酶解75min。通过原生质体诱变筛选,得到1株高产菌株,磷脂酶D水解活力达4.29 U/m L,提高幅度为180.4%。该方法有效改善了链霉菌野生菌株原生质体的制备效果,紫外诱变筛选显著提高了磷脂酶D的活力,高产突变株具有较好的稳定性。     

阿维链霉菌的诱变育种

以阿维链霉菌(Streptomyces avermitilis)76-12为出发菌株,采用亚硝基胍、吖啶橙、紫外线和氯化锂分别对其孢子和原生质体进行诱变,经抗代谢物理性筛选,获得一系列高产突变株,其中N-1-2高产突变株的发酵单位是出发菌株的2.47倍。实验中同时获得了只产阿维菌素a组分的突变株G-32、Bla组分含量高的Ave8菌株和产蓝绿色孢子的突变株UA-G等。     

亚硝基胍消除链霉菌FR-008的线性质粒

【目的】利用亚硝基胍(NTG)消除链霉菌FR-008线性质粒以简化其基因组,获得背景清晰的菌株,作为抗生素异源生物合成的底盘细胞。【方法】NTG溶液处理链霉菌FR-008孢子悬液,从存活的诱变株中筛选砷敏感的突变株,再通过脉冲场凝胶电泳(PFGE)检测线性质粒是否被消除;用生测实验定性检测各个线性质粒消除突变株杀念菌素合成的能力,最后通过HPLC定量比较突变株和野生型产生杀念菌素的差异。【结果】从103个诱变株中筛选到3株砷敏感的突变株(10#、59#、115#)。PFGE检测发现它们均丢失了大线性质粒p SSFR1,此外,42#突变株的小线性质粒p SSFR2被消除,在此基础上,第二轮NTG诱变获得了双质粒消除的突变株。大线性质粒p SSFR1消除率约为3%,小线性质粒p SSFR2消除率约为1%。发酵结果显示:10#、115#突变株杀念菌素有效组分III产量分别提高了40%和30%。【结论】首次发现NTG是一种有效消除链霉菌线性质粒的诱变剂,2株大线性质粒消除的突变株杀念菌素的产量得到提高。此方法可以用来消除特定链霉菌菌株中的巨型线性质粒以高效简化其基因组,因而是一种有效的抗生素遗传育种的方法。     

阿维菌素B1a组分高产菌株的定向选育

以阿维链霉菌(Streptomyces avermitilis)1-17为出发菌株,分别使用紫外线及亚硝基胍并结合L-异亮氨酸诱导手段进行诱变处理,得到AVMB1a组分摇瓶发酵水平较出发菌株提高12.86%的突变株3-6.传代实验表明该菌株的高产性能稳定.结果表明,采用UV、NTG诱变结合L-Ile诱导的手段可以获得B1a组分显著提高的菌株.     

紫外诱变选育恩拉霉素高产菌株

本研究采用紫外诱变育种技术对一株产恩拉霉素抗真菌链霉菌(Streptomyces fungicidicus)F110进行了诱变处理,经链霉素抗性、利福霉素B抗性以及双重抗性筛选,共获得了132株抗生素抗性突变株,其中26株突变菌株的恩拉霉素产量与出发菌株相比均有明显提高。摇瓶发酵条件下,突变株SR93的恩拉霉素产量最高可达2 400μg/m L,与出发菌株相比提高了38%。传代结果表明:该突变株产素水平稳定,因此具备较好的开发及工业应用价值。     

链霉菌702产孢子固体培养基和培养条件的筛选

通过单因素实验、均匀设计和正交实验对链霉菌702产抱子培养基和培养条件进行筛选,筛选到的最佳培养基组成为：马铃薯2009／L,葡萄糖25g／L．最佳培养条件为培养温度37℃、培养时间5天和培养基初始pH值8．实验结果表明,采用优化后的培养基和培养条件,使链霉菌702产抱子数达到4．07亿,比原来培养基（高氏一号培养基）产抱予量提高了8．7倍,培养时间却从原来的7天缩短到现在的5天．     

The tsr gene-coding plasmid pIJ702 prevents thiopeptin from inhibiting ppGpp synthesis in Streptomyces lividans

Streptomyces lividans normally accumulated high levels of ppGpp during nutritional shift-down. Its accumulation was, however, severely inhibited when a small amount of thiopeptin (an analogue of thiostrepton) was included in the transfer medium. In contrast, a S. lividans strain, which harbours the plasmid pIJ702 carrying the tsr gene resist to thiopeptin through methylation of the 23S rRNA, was still capable of accumulating ppGpp in the presence of large amounts of thiopeptin. These results indicate that the rRNA methylation resulting from the action of tsr gene prevents thiopeptin not only from inhibiting cell-growth but also from inhibiting ppGpp synthesis. The results also indicate that the observed accumulation of ppGpp during nutritional shift-down was associated with ribosomal function, as has been shown in E. coli and B. subtilis.     

红谷霉素生料发酵工艺的研究

红谷霉素是链霉菌702发酵所产的一种生物活性物质,具有较强抗细菌活性。在摇瓶条件下,对链霉菌702生料发酵生产红谷霉素进行研究。采用单因素实验探索生料发酵工艺,筛选出摇瓶生料发酵条件为7.5%的种子接种量和添加抗菌剂1.5ml。在此条件下进行生料发酵,使红谷霉素摇瓶发酵产量达到1.46g/L,比常规灭菌发酵产量提高25.7%,实验结果表明红谷霉素生料发酵是一种潜在可行的发酵方法,不但可以很好的节约发酵成本,还可以提高发酵产量。     

Detection of gentoxicity of benzidine and its derivatives with the Escherichia coli DJ 702 lacZ reversion mutagenicity assay

AIMS: The feasibility of Escherichia coli DJ 702 lacZ mutagenicity assay to detect genotoxicity of benzidine and its derivatives was evaluated. METHODS AND RESULTS: DJ 702 strain was grown overnight at 30 degrees C in Luria-Bertani (LB) medium containing some components, such as chloramphenicol, ampicillin, delta-aminolevulinic acid, isopropyl-beta-d-thiogalactoside, and trace element mix. The mixtures of a bacterial culture and tested chemical at indicated doses were incubated at 30 degrees C for 30 min. Subsequently, 2 ml of molten top agar was added and the resulting mixtures were immediately poured onto a minimal lactose (ML) plate. Plates were incubated at 30 degrees C for 48 h. The number of colonies was determined by visual scoring. In this study, results showed that all the tested chemicals were mutagenic to DJ 702 strain. CONCLUSIONS: E. coli lac mutagenicity assay using DJ 702 strain can detect the mutagenicity of benzidine and its derivatives. SIGNIFICANCE AND IMPACT OF THE STUDY: We detected the mutagenicity of benzidine and its derivatives in E. coli lac mutagenicity assay using DJ 702, indicating that this assay may be used to detect benzidine and its derivatives in a powerful, sensitive, and convenient mutagenesis assay.     

链霉菌LA5高产菌株诱变育种研究初报

抗生素LA5具有开发成抗真菌农用抗生素的广阔前景,但由于该菌株的野生型菌株发酵效价低,不能满足工业化开发的要求。通过以链霉菌LA5为出发菌株,采用紫外线(UV)、微波、亚硝基胍(NTG)、紫外线 亚硝基胍、亚硝基胍 紫外线等方法进行诱变筛选,结果表明,以紫外线照射80 s 亚硝基胍处理80 min的诱变的正突变率最高,为30%,其他依次为亚硝基胍处理80 min 紫外线照射40 s诱变和亚硝基胍处理60min 紫外线照射120 s诱变,正突变率均为26%。然后依次使用亚硝基胍处理80 min 紫外线照射80 s、亚硝基胍处理80 min 紫外线照射40 s、亚硝基胍处理60 min 紫外线照射120 s进行第2、第3、第4轮复合诱变筛选,最后选育出突变菌株U8-N8A-196,其产抗生素能力比链霉菌LA5出发菌株提高了34.5%,极显著高于LA5菌株。     

大孔吸附树脂吸附链霉菌702抗真菌活性物质工艺

研究大孔吸附树脂吸附链霉菌702抗真菌活性物质的工艺条件。采用5种不同大孔吸附树脂吸附链霉菌702发酵液提取液中抗真菌活性物质,选择吸附效果较佳树脂进行吸附条件优化,以桔青霉为指示菌,纳他霉素为对照抗生素．采用“管碟法”测定抗真菌活性物质含量。结果发现,XAD18树脂吸附效果较好,获得优化吸附条件：上样液pH6,NaCl质量浓度10g/L,上样量22．63mg／g湿树脂,吸附流速2．5mL／min,水洗体积180mL,洗脱流速1．5mL／min,洗脱剂为体积分数10％、50％和90％的甲醇,洗脱方式为梯度洗脱。在确定的工艺条件下XAD18对链霉菌702抗真菌活性物质的吸附率可达90％,洗脱率可达75％,回收率可达80％。     

Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702

The cholesterol oxidase gene (cho) of Streptomyces sp. was cloned into Streptomyces lividans with the vector pIJ702. Deletion analysis of the recombinant plasmid showed that entire coding sequence of the cho gene was located within a 2.5-kilobase segment of the chromosomal DNA obtained from the cholesterol oxidase-producing strain. When cloned cells of S. lividans were grown in an appropriate medium, the cells produced severalfold more cholesterol oxidase extracellularly than did the producing strain.     

Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702.

The cholesterol oxidase gene (cho) of Streptomyces sp. was cloned into Streptomyces lividans with the vector pIJ702. Deletion analysis of the recombinant plasmid showed that entire coding sequence of the cho gene was located within a 2.5-kilobase segment of the chromosomal DNA obtained from the cholesterol oxidase-producing strain. When cloned cells of S. lividans were grown in an appropriate medium, the cells produced severalfold more cholesterol oxidase extracellularly than did the producing strain.