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1、工业微生物菌种选育技术,链霉菌基因工程,为什么链霉菌？,链霉菌的重要性,产生绝大多数已知的抗生素 生物合成机制的阐明 生产工业用酶的潜力 葡萄糖异构酶 蛋白酶 纤维素酶 木聚糖酶 。,What Are Streptomyces?,They are Gram Positive filamentous Bacteria. They are obligate aerobic bacteria that are abundant in most soils. Streptomyces have two metabolic stages, Primary and Secondary Metabolism
2、. They utilize insoluble organic debris by producing a variety of extracellular hydrolytic enzymes.,放线菌生活史,Streptomyces coelicolor colonies with aerial mycelium and spores,The area of the picture represents about 2x3 cm. The blue haloes around the colonies are secreted actinorhodin. Actinorhodin is
3、an antibiotic (not used clinically) which is blue under alkaline conditions and red under acidic conditions. Actinorhodin is a polyketide made by multiple condensations of acetate by a Type II polyketide synthase (John Innes Centre, Photography Department).,Young vegetative mycelium,Young vegetative
4、 mycelium (c. 1 m wide) of Streptomyces grown in liquid broth (light microscope ; Gabriella Kelemen John Innes Centre).,Mutant colonies of Streptomyces coelicolor,c. 1 cm x 1 cm. The wild-type colonies are covered with grey aerial mycelium and spores; the reddish mutant colonies are not forming aeri
5、al mycelium. The red mycelium colour and the dark background is from the antibiotics produced by Streptomyces coelicolor (John Innes Centre, Photography Department).,Single colony of Streptomyces coelicolor,Single colony of Streptomyces coelicolor topped by a liquid droplet containing antbiotic. Liq
6、uid droplets are often found in Streptomyces colonies, it is not clear how they are generated. (John Innes Centre, Photography Department).,Close up of a colony of Streptomyces coelicolor mutant D132,Close up of a colony of Streptomyces coelicolor mutant D132 which is produces undecylprodigiosin but
7、 not actinorhodin (image provided by Karen Jolly, University of Leeds).,Mass of curled aerial mycelium of Streptomyces coelicolor,Mass of curled aerial mycelium (c. 1 m wide) of Streptomyces coelicolor . (Scanning electron micrograph, Mark Buttner, Kim Findlay, John Innes Centre).,Aerial mycelium an
8、d spore of Streptomyces coelicolor,The mycelium and the oval spores are about 1m wide, typical for bacteria and much smaller than fungal hyphae and spores. (Scanning electron micrograph, Mark Buttner, Kim Findlay, John Innes Centre).,Close up of Streptomyces coelicolor spores,Close up of Streptomyce
9、s coelicolor spores (c. 1m wide) showing surface structures. (Scanning electron micrograph, Mark Buttner, Kim Findlay, John Innes Centre).,What Are Antibiotics?,Antibiotics are compounds that are products of secondary metabolism. Antibiotics can range from 100 to about 1200 in molecular weight. Thes
10、e compound inhibit the growth of other competitive microbes by interfering in their normal biochemistry. Of the thousands of antibiotics that have been discovered, two-thirds are produced by Streptomyces.,Chronological table of novel antibiotic discovery,链霉菌宿主菌的条件,遗传背景清楚，不带内生质粒； 由于质粒的转化主要借助原生质体，因此宿主
11、菌的原生质体制备和再生必须易于进行； 宿主菌相对于载体上的标记必须是“缺陷”的(即对标记的抗生素敏感，对产色素标记缺陷)； 宿主菌必须没有限制作用。,链霉菌宿主菌,Streptomyces lividans TK1326、TK24、TK64等 Streptomyces coelicolor A3(2) Other Streptomyces,Vectors for Streptomyces,高拷贝载体:pIJ101, pIJ702 低拷贝载体:pIJ61, pIJ922, pIJ941 穿梭载体: pIJ699 Cosmid:pKC505 接合转移载体:pPM801 噬菌体载体:fC31系列 表
12、达载体:pIJ6021, pIJ4123 分泌载体,Other vectors,大容量载体：采用酵母人工染色体（YAC）克隆技术构建了细菌BAC载体，插入片段100300kb。 整合型载体：pSAM2， pHZ808 高表达载体：pCJR24, pCJR29,常用的链霉菌质粒载体,质粒pIJ702,链霉菌基因转移方法,原生质体转化 接合转移 电穿孔转移（Electroporation） 噬菌体转染与转导,克服宿主限制性修饰系统,甲基化缺陷的大肠杆菌宿主 用lividans作为过渡 PCR获得DNA 单链DNA 原生质体热处理 体外去除甲基化修饰 突变株宿主,链霉菌质粒转化,转化的注意点,PEG
13、的影响：批号、分子量、浓度 R2YE平板的干燥度：15%左右 加抗生素筛选的时间,Protocol for transformation of Streptomyces by electroporation,接合转移的优点,免除原生质体制备、再生 降低限制修饰作用 完善的质粒载体 穿梭质粒 pSET152, oriT,接合转移,oriT质粒转化ET12567,抗性转化子培养过夜,稀释后培养至A6000.5,LB洗涤，以0.1倍悬浮,108孢子50热激10分钟,等体积混合大肠杆菌和链霉菌孢子,混合涂布，萘啶酮酸、抗生素筛选,菌丝体也适用,克隆次级代谢途径生物合成基因的方法,抗生素生物合成基因克隆
14、策略,将目的基因克隆到标准宿主菌，然后检测个别基因产物。 克隆与阻断突变株互补的基因。 利用突变克隆，分析生物合成基因簇（gene cluster）。 克隆抗性基因并分析连锁的生物合成基因,克隆策略续,用人工合成的寡核苷酸探针探测基因文库 直接克隆抗生素产生菌的DNA大片段到非抗生素产生菌中，检测产物的表达。 利用已克隆的生物合成基因为探针克隆同源基因。 PCR扩增已知基因。,将目的基因克隆到标准宿主菌，然后检测个别基因产物,Target gene:灰色链霉菌中与杀念菌素生物合成有关的对氨基苯甲酸(PABA)合成酶基因pab。 宿主和载体：变青链霉菌，BamHI片段插入到pIJ41的BamHI
15、位点 筛选方法与结果： 以过量产生PABA的灰色链霉菌的磺胺抗性株为供体，在硫链丝菌素抗性转化子中选择磺胺抗性克隆子； 以野生型为供体，pab营养缺陷型为受体选择原养型克隆子。结果从两种选择方法中各获得一个克隆子，每个克隆都带有45kb的BamHI片段,方法1续,Target gene:抗生链霉菌中与放线菌素生物合成有关的吩噁嗪酮(phenoxazinone)合成酶(PHS)基因。 宿主和载体：变青链霉菌66，SphI片段克隆到pIJ702的SphI位点上。 筛选方法与结果：从5000个硫链丝菌素的抗性转化子中检测从3-羟基蒽林酸产生黄色朱红菌酸的转化子，获得了三个克隆子，其中一个携带了编码P
16、HS结构基因的2.45kb的SphI DNA片段，另外两个各含有1.77kb和4.28kb的SphI片段。当转入变青链霉菌后，起激活变青链霉菌染色体上沉默基因的作用。 本方法的优点是不需要了解抗生素生物合成途径的遗传信息，但要知道何种酶参与了生物合成以及具有检测这种酶的灵敏的分析方法。,克隆与阻断突变株互补的基因,抗生素的生物合成途径通常涉及到1030酶反应步骤，如果通过诱变的方法使其中的一个酶失活，而使其不能合成最终产物，这种突变株称为阻断突变株（Blocked mutants）。如果克隆了一段外源基因而使抗生素的合成恢复，表明这段基因互补了原先失活的酶基因 Target gene: 天蓝色链霉菌中十一烷基灵菌红素(Red)生物合成基因。Blocked Mutants：首先用紫外光诱变天蓝色链霉菌，经筛选获得37个不产Red的突变株，通过共合成试验将突变株分成5类(red AE)，每组的代表已在天蓝色链霉菌染色体上作出了一簇紧密连锁的位点图。 宿主和载体：阻断突变株red E60，将天蓝色链霉菌的 DNA用BclI和PstI完全酶解或Mbo
