链霉素生产菌—灰色链霉菌和热灰紫链霉菌的原生质体融合的研究

本文采用原生质体融合技术,把链霉素生产菌——灰色链霉菌No．45 Streptomyces griseus No.45(Lin,Rif‘)同耐高温的不产生抗生素的热灰紫链霉菌T272 Strep-griseus thermogriseoviolaceus T272(Lin^s,Rif^f)进行了原生质体融合。以抗性为选择标记,以PEG6000为助融剂,选出了融合体。制备超薄切片后在电镜下观察了原生质体融合的详细过程。在链霉素生物合成受抑制的高温(37℃)下,测定了融合体的抑菌括性,从形态上与两亲株不同的46株融合体中发现6．3％的融合体既有耐高温的特性也有抑菌的活性。     

庆大霉素产生菌棘孢小单孢菌原生质体形成、再生及融合重组的研究

在含0．3％甘氨酸的液体培养基中培养庆大霉素产生菌——棘孢小单孢菌,其菌丝形态有明显的改变,易被溶菌酶消化细胞壁而释放大量原生质体。菌丝的培养龄影响其相应原生质体的再生活性(即再生成菌落的能力)。以72h为最佳,48及120h菌龄的原生质体再生频率依次相当于72h的40％和10％左右。以PEG4000为融合剂。用直接法检出重组子,重组频率约为10～6,融合重组子中有一些菌株庆大霉素的摇瓶发酵效价在1900u／m1左右,比对照菌株有很大的提高。     

电场诱导棘孢小单胞菌原生质体融合

以小诺霉素（Micromomicin,MCR)产生菌棘孢小单胞菌（Micromonosporaechinospora）为出发菌株,诱变筛选得到一株链霉素抗性菌株,抗性菌株的原生质体分别用紫外线照射和热处理致死,通过单亲致死原生质体融合,以链霉素为选择条件选出融合株,经摇瓶发酵并结合薄层层析扫描（ThinLayerCharamotography,TLC）分析,筛得4株MCR百分含量高于亲株的融合子,MCR百分含量达到90％,效价1076u／ml,分别比亲株提高15％和11％。     

林肯链霉菌双亲灭活原生质体融合的研究

分别以紫外线、热灭活林肯链霉菌 94 7和 95 0 2原生质体 ,然后进行灭活双亲的原生质体融合 ,从 1 6株融合子筛选到林肯霉素高产株。用双亲的互补营养缺陷型对林肯链霉菌原生质体的制备、融合、再生的部分条件进行了研究。发现含 0 .4 %Gly和 34 %蔗糖的SM培养基最适于实验菌株原生质体的制备、再生。聚乙二醇 (PEG)分子量对原生质体融合影响不大 ,其在P缓冲液中的浓度却很重要。含 5 0 %PEG的P缓冲液最有利于原生质体融合     

吸水链霉菌应城变种和庆丰链霉菌种间原生质体融合及其融合子的生物学特性

本文报道利用抗生素产生菌吸水链霉菌应城变种Leu^-Sm^R。90-11菌株(Leu’smr)和庆丰链霉菌A_553-1菌株(Pro^-Sm^R)为亲株,以42％的PEG4000为助融剂,进行了种问原生质体融合,用间接法检出融合重组子38株,其重组频率约为4．5×10^-4。重组子除孢子丝形态外,孢子堆颜色、抗药性、抗菌活性和抗生素生物合成能力方面与亲株均有一定差别,而且不同重组子之间也不相同,特别在抗菌活性方面,其中重组子FL-42和FL-48不仅具有两个亲株所产生的4种抗生素的活性,而且还产生两个亲株不具有的活性物质。通过纸层析谱表明,这种活性物质,两个重组子之间也不相同。     

链霉菌原生质体的再生

（续１９９８年第３３卷第１期第４１页）３原生质体的再生链霉菌原生质体的再生是指经去壁的原生质体在高渗的再生培养基上,一方面再生出原有的细胞壁,恢复菌丝原来的完整形态,另一方面恢复细胞的生理功能,保证细胞萌发、生长和分裂。原生质体再生过程有以下几个步骤...     

链霉菌原生质体的制备

原生质体是细胞去除了坚韧细胞壁后对渗透压极为敏感的球质体,最外层是裸露的细胞质膜,失去了细胞壁的原生质体,染色体ＤＮＡ在诱变剂作用下,更易引起死亡突变,敏感性提高。菌种的敏感性越高,诱发突变的机会越大。因此,用原生质体代替孢子、菌丝细胞作为诱变育种的...     

天然无抗菌活性链霉菌种间原生质体融合与活性重组体的分离

天然无抗菌活性的变青链霉菌1326与链霉菌1254的营养缺陷型变株进行种间原生质体融合,获得了原养型融合体,频率为10^-6—10^-5。融合前双亲株原生质体经52℃水浴处理4min,融台频率有所提高,约有一半的融合体菌株在琼脂平板上培养后显示有不同程度的抗菌活性,但绝大多数皆不稳定。从755株融合体中筛选到5株抗菌活性较稳定的菌株,并对其中三株所产生的抗生素作了初步鉴别。发现一株产生中性脂溶性抗生素,其余两株所产生的抗生素皆为碱性非典型脂溶性物质。尽管此等产物的理化性质还有待进一步鉴别,从中发现新化合物的几率是否大于传统的筛选途径,也还需要更多的筛选实践才能说明,但为了进一步开发利用微生物资源,这一途径似值得探讨。     

卡奇霉素产生菌棘孢小单胞菌ATCC 15837的全基因组测序及序列分析

棘孢小单胞菌(Micromonospora echinospora) ATCC 15837是一种高GC含量的革兰氏阳性稀有放线菌,能够合成烯二炔类抗肿瘤抗生素卡奇霉素(calicheamicin, CLM)。目前,还没有相关研究报道棘孢小单胞菌ATCC 15837的全基因组序列,这限制了其代谢产物合成途径和比较基因组学等研究。本研究首次通过高通量测序技术对棘孢小单胞菌ATCC 15837进行全基因组测序,使用相关生物信息学软件对数据进行组装和注释等分析。使用Velvet软件进行组装拼接得到77个Contigs,GC含量为72.36%,基因组大小约为7.69 Mb。序列已提交至美国国立生物技术信息中心(NCBI)的GenBank数据库(登录号为NGNT00000000)。本研究首次报道了一株烯二炔类抗肿瘤抗生素卡奇霉素产生菌棘孢小单胞菌ATCC 15837的全基因组序列,分析了基因组基本特征,预测了该菌株的次级代谢产物生物合成基因簇,为后续的进一步代谢调控与合成生物学提供了理论基础。     

灰色链霉菌胰蛋白酶在变铅青链霉菌中的异源表达及酶学性质分析

胰蛋白酶作为一种重要的丝氨酸蛋白酶被广泛应用于食品、医药和皮革等工业领域.本文成功实现了灰色链霉菌来源的胰蛋白编码基因在变铅青链霉菌中的高效活性表达,并对其酶学性质进行分析比较.以灰色链霉菌ATCC10137基因组为模板,获得胰蛋白酶编码基因sprT并克隆至表达质粒pIJ86,成功构建了重组链霉菌工程菌TK24/pIJ86-sprT.以R2YE和SELF为发酵培养基,最高酶活分别达9.21 U/mL和8.61 U/mL.酶学性质分析表明,和牛胰蛋白酶(BT)相比,重组链霉菌胰蛋白酶(rSGT)的耐酸能力强,具有较广的pH;且rSGT对酰胺键具有更高的特异性;此外,Zn2+和有机溶剂分别对rSGT的酯酶活力和酰胺酶活力具有促进作用;本研究结果为rSGT的性质改造以及工业应用提供了依据.     

S-adenosylmethionine activates adpA transcription and promotes streptomycin biosynthesis in Streptomyces griseus

Using a two-plasmid system, we recently identified sigma(E)-dependent promoters directing expression of the sigma(E) regulon genes in Salmonella enterica serovar Typhimurium (S. Typhimurium). Comparison of the promoters revealed a consensus sequence almost identical to the sigma(E)-dependent rpoEp3 promoter directing expression of rpoE. This two-plasmid system was previously optimized to identify nucleotides critical for the rpoEp3 promoter activity. However, two highly conserved nucleotides in the sigma(E) consensus sequence were not identified by this screening. In the present study, we have improved the two-plasmid screening system using a new optimized error-prone PCR mutagenesis. Together with site-directed mutagenesis, we further identified nucleotides critical for activity of the rpoEp3 promoter and quantified the effect of the particular mutation upon promoter activity. All the identified critical nucleotides of the rpoEp3 promoter (in capital) were located in the -35 (ggAACtt) and -10 (gTCtaA) regions and corresponded to the most conserved nucleotides in the sigma(E) consensus sequence. The expression of the wild-type and mutated rpoEp3 promoters was confirmed in S. Typhimurium and was found to exhibit a different pattern of sigma(E) activation compared with Escherichia coli, with a peak rpoEp3 promoter activity in early stationary phase followed by a decrease in late stationary phase.     

Transformation using in vivo and in vitro methylation in Streptomyces griseus

Streptomyces griseus does not readily take up foreign DNA isolated from other Streptomyces species or Escherichia coli, presumably due to its unique restriction-modification systems that function as a barrier for interspecific DNA transfer. To efficiently transform S. griseus by avoiding the restriction barriers, we methylated incoming DNA in vivo and in vitro and treated protoplasts with heat prior to transformation. Whereas heat treatment of protoplasts or methylation of the E. coli-Streptomyces shuttle vectors (pXE4 and pKK1443) did not prominently improve the transformation efficiency, HpaII methylation of the vectors from any E. coli strains tested in this study highly increased the transformation efficiency. The highest transformation efficiency was observed when the shuttle vectors were isolated from the dam, hsd strain of E. coli (GM161) and methylated by AluI and HpaII methyltransferases, and the efficiency was approximately the same as that of the vectors from S. griseus. We identified several restriction-modification systems that decrease the transformation efficiency. This research also led us to understand methylation profiles and restriction-modification systems in S. griseus.     

In vitro protein phosphorylation associated with cellular differentiation of Streptomyces griseus

In vitro phosphorylation reactions with crude cellular extracts revealed that phosphorylation of a 17-kDa protein is associated with the onset of aerial mycelium formation in solid culture (but not submerged spore formation in liquid culture) of Streptomyces griseus. The possible importance of the 17-kDa protein phosphorylation in cellular differentiation was further indicated by inducing aerial mycelium formation in the presence of decoyinine and in studies using certain developmental mutants (relC, afsA, and M-1). It is proposed that the 17-kDa protein may play a role in cellular differentiation of S. griseus via its phosphorylation. Received: 17 July 1997 / Accepted: 20 September 1997     

响应面法优化灰色链霉菌产纤维素酶的工艺研究

以灰色链霉菌为原料,在单因素试验的基础上,采用响应面法试验,优化灰色链霉菌产纤维素酶活性的发酵条件。结果表明,单因素试验灰色链霉菌产纤维素酶活性的最适发酵条件:碳源为CMC-Na,氮源为明胶,温度为28℃,pH为7.0,转速为130 r/min。响应面法试验优化灰色链霉菌产纤维素酶活性最佳发酵条件为:温度27.7℃,pH值6.9,转数130.3 r/min,在此优化条件下,灰色链霉菌产纤维素平均酶活性为6.103 U/mL(n=3),与模型的预测值(6.217 U/mL)比较接近,误差为1.83%,证明了该响应面模型具有可靠性。     

Proteins that interact with GTP in Streptomyces griseus and its possible implication in morphogenesis

Abstract By cross-linking with [α-³²P]GTP or [γ-³²P]GTP with or without UV treatment, several proteins of Streptomyces griseus were shown to interact with GTP in specific ways. After gel electrophoresis, 19 bands of radioactivity were found; 12 bands were assigned as GTP-binding proteins and 6 bands as phosphorylated proteins. One band was assumed to be a guanylylated protein. The profile of radioactive bands was similar between cells prepared from liquid or solid culture, but markedly different between growth phases. A mutant (strain M-1) defective in aerial mycelium formation, which was originally found as a decoyinine-resistant isolate, was found to have a different profile of phosphorylated proteins.     

灰色链霉菌启动子活性片段在大肠杆菌中的亚克隆及其序列分析

通过对重组质粒No.8-1的亚克隆,灰色链霉菌插入到大肠杆菌载体质粒中的序列已缩小到410bp,仍具有启动子功能。序列分析表明,启动子活性片段的G C碱基组成为50.5%。内含链霉菌启动子区域常有的正向重复序列;有1个Alul位点,1个Clal位点,2个Mbol位点,3个Nla Ⅲ位点,1个Pvu Ⅱ位点;具有类似于E.coli启动子的保守序列-10区和-35区,两者间隔18bp;在相应位置上分别有一段序列与E.coli的SD序列和在苄铅青链霉菌的SEP(Streptomyces-E.coil-type promoter)序列中存在的保守序列具有一定的相似性。     

秦岭链霉菌的原生质体再生与诱变育种

在秦岭链霉菌(Streptomyces qinlingensis sp. nov.)的菌种改良中, 应用原生质体再生并结合物理化学诱变能够得到产量较高、稳定性较好的菌株。筛选实验表明：秦岭链霉菌原生质体再生菌株R-72、诱变菌株NTG-1和H30-7对枯草芽孢杆菌的抗菌活性均提高了20%以上, 并且连续培养10代, 其遗传性状均比较稳定。进一步的生测实验表明菌株R-72、NTG-1和H30-7对5种病原细菌和5种植物病原真菌的抗菌活性相比原始菌株有显著提高。     

秦岭链霉菌的原生质体再生与诱变育种

在秦岭链霉菌(Streptomyces qinlingensis sp.nov.)的菌种改良中,应用原生质体再生并结合物理化学诱变能够得到产量较高、稳定性较好的菌株.筛选实验表明:秦岭链霉菌原生质体再生菌株R-72、诱变菌株NTG-1和H30-7对枯草芽孢杆菌的抗菌活性均提高了20%以上,并且连续培养10代,其遗传性状均比较稳定.进一步的生测实验表明菌株R-72,NTG-1和H30-7对5种病原细菌和5种植物病原真菌的抗菌活性相比原始菌株有显著提高.