应用双亲株灭活原生质体融合法选育林可霉素高产菌株

目的：选育高产优质林可霉素产生菌。方法：将林可霉素产生菌S78－1^#,N65-2^#的原生质体分别热灭活和紫外灭活，并将两种灭活原生质体用PEG融合，从融合株中筛选高产优质菌种。结果：获得82－201^#融合株，其摇瓶效价分别较N65－2^#,S78-2^#提高17％和52％，且色素分泌少，传代稳定性强。结论：双亲株灭活原生质体融合选育林可霉素高产菌株是值得推广的一种选育方法。     

地中海拟无枝酸菌原生质体电融合及其在提高利福霉素SV发酵效价中的应用

对产利福霉素SV的地中海拟无枝酸菌 (Amycolatopsismediterranei)原生质体进行热灭活和紫外灭活标记 ,其热灭活条件为 5 2℃ ,1.5h ,紫外灭活条件为紫外线照射 ( 15W ,2 5cm ,2 70nm) 6 0min。将双灭活标记的原生质体用CRY 3型细胞融合仪进行电融合 ,得出 :成串电流频率 1.5MHz ,电压 5 8V ,成串时间 2 0s ,融合脉冲幅宽 80 μS ,融合电压 5 0 0V ,融合槽间距 0 .5mm时 ,融合率最高为 9.3× 10 -4 。对筛选的融合子产量试验表明 ,5 0 %以上的融合子产量高于出发菌株 ,有明显的正变作用 ,将融合子在利福霉素代谢类似物平板上分离纯化 ,获得了化学效价提高 30 %以上的产量稳定菌株。     

非对称灭活原生质体融合法选育头孢菌素C高产菌株

目的选育高产头孢菌素C产生菌。方法分别以紫外线和加热灭活头孢菌素C产生菌Cephalosporium acremonium 18-U5和Cephalosporium acremonium 18-M6原生质体,并将两种灭活的原生质体经PEG4000融合,从融合株中筛选头孢菌素C高产菌株。结果获得高产头孢菌素C的顶头孢霉融合株Cephalosporium acremonium F20,发酵单位达4 655 mg.L-1。与双亲菌株相比分别提高了42.4%和34.9%。结论非对称灭活原生质体融合法选育头孢菌素C高产菌株的方法值得推广。     

双亲灭活原生质体融合法选育阿维菌素高产菌株

目的选育高产阿维菌素产生菌。方法分别以紫外线和加热灭活阿维菌素产生菌Strepto-myces avermitilis620和Streptomyces avermitilis632原生质体,并将2种灭活的原生质体用PEG4000融合,从融合株中筛选阿维菌素高产菌种。结果获得高产阿维菌素融合株StreptomycesavermitilisF32,总发酵单位达3 904 mg.L-1,其中B1a组分产量较高,达1 016 mg.L-1,分别较出发菌株S.avermitilis 620、S.avermitilis 632提高117.1%和103.6%。结论双亲灭活原生质体融合法选育阿维菌素高产菌株是值得推广的一种选育方法。     

微生物原生质体灭活及其在育种中的应用

灭活原生质体融合技术的依据是,用适当的方法处理单一亲株或双亲株的原生质体,使之失去再生的能力,经细胞融合后,由于致死损伤部位的互补可以形成能再生的融合体,除有诱变作用的灭活剂外,一般的灭活处理对细胞DNA的遗传功能和重组力未发现有明显的影响。该技术在动、植物细胞融合中应用较多,近几年来在微生物研究中的应用亦有较大发展。一、原生质体灭活实验技术主要采用热、紫外线(UV)、电离辐射(X-射线、γ-射线)以及某些生化试剂(包括抗生素)等作为灭活剂。 (一)热灭活通常用50-52℃处理2h。在处理过程中,可将原生质体悬液用移液管移至预热过的高渗琼脂培养基表面     

林可链霉菌与委内瑞拉链霉菌种间原生质体融合的研究

本文报道氯霉素产生菌委内瑞拉链霉菌A-186株与林可霉素产生菌林可链霉菌林可变种78-11株种间原生质体融合的结果。首先,研究了委内瑞拉链霉菌A-186的原生质体制备和再生条件,再生率可达86％。然后,采用氯霉素产生菌S.venezuelae A-186的原生质体用紫外线灭活后,与具有耐药标记的S.lincolnensis var.lincolnensis78-11原生质体融合的方法,种间融合频率达到3.2×10~(-5) 。并筛选到一株遗传稳定的重组子,经初步鉴定它能同时产生两亲株所产的两种抗生素。     

不同浓度过氧乙酸灭活HBV效果观察

本文通过对0．2％0．3％0．4％……1．1％十种不同浓度的过氧乙酸作用不同时间，观察其对HBV灭活效果，发现有三点与文献报告有所不同：①HBsAg抗原性的灭活时间延长：0．2％180分钟、0．3％120分钟、0．4％60分钟、0．5％60分钟、0．6％30分钟，浓度在0．7％以上者5分钟内即可灭活。②HBV—DNA灭活所需过氧乙酸浓度降低及时间缩短，0．2％5分钟不能灭活HBV—DNA，超过5分钟或浓度大于0．2％者5分钟内即可灭活HBV—DNA。③HBsAg抗原性的消失与HBV—DNA活性消失存在一定距离对BSAg抵抗化学因子较HBV—DNA强，破坏HBsAg抗原性较HBV－DNA难，提示在观察和检测消毒剂灭活HBV效果时应同时观察HBsAg、HBV－DNA，在实际操作中，应加大消毒剂浓度或延长消毒时间，以快速彻底地杀灭HBV。     

青霉素产生菌的原生质体融合

将细菌血红蛋白基因引入青霉素产生菌产黄青霉H－106中而获得了产黄青霉基因工程菌1M5。产黄青霉20^#是一个青霉素产量高但产孢子能力偏低的生产菌。为提高产黄青霉的产量及产孢子能力，降低产黄青霉对氧的需求，我们采用原生质体融合技术，将通过UV诱变处理的检出的产黄青霉基因工程菌1M5 Met^-缺陷型菌株和产黄青霉20^#Arg^-缺陷型菌株分别用Novozyme 234和Cellulase R-10(1∶1）混合酶处理所得原生质体按1∶1混合后加入30％聚乙醇6000融合，产物分离纯化，得到融合株C－396^#菌株，其青霉素产量和产孢子能力都有所提高，对氧的需求有所降低。     

黑暗链霉菌原生质体融合

报道了黑暗链霉菌原生质体制备和融合的方法。将一原养型亲株原生质体经紫外线灭活2分钟,与另一苯丙氨酸缺陷型突变株原生质体等量混合,40％PEG处理,32℃保温3分钟,获得了重组后代,经分析,融合子的代谢产物中次组分的含量有所降低。     

拟无枝酸菌B37与游动放线菌E92—70属间隔合

以去甲基万古霉素产生菌Ｂ３７和替考拉宁类抗生素产生菌Ｅ９２－７０为出发菌株，将Ｂ３７原生质体热灭活，进行属间原生质体融合，融合率１．４×１０＾－６－４．６×１０＾－６，融合子５０％以上抗菌活性增强。     

林可霉素生物合成基因lmbH的回转化

以ＰＩＪ７０２或ＰＩＪ４８６为载体构建了３个含有ｌｍｂＨ基因的重组质粒ｐＥＳＳ７０１、ｐＥＳＳ７０４和ｐＥＳＳ７０８分子,分别回转化林可霉素产生菌Ｂ４８,并随机挑选了６００个回转化克隆进行摇瓶初筛,以抑菌圈直长作为稀理林可霉素产生量的标准。     

林可霉素高产菌种的选育

以林可霉素产生菌82－7^#为出发菌株,采用氯化锂、紫外线（uV)、甲基磺酸乙酯（EMS）三重复合诱变处理、筛选耐自身产物的高产菌种,从中获得65－12^#菌株。其摇瓶效价较出发菌株提高25％,发酵培养基优化组合后,其摇瓶效价提高28％。应用于30吨发酵罐生产,其发酵指数较出发菌株提高16．8％。     

纳他霉素产生菌基因组重排育种

Streptomyces gilvosporeus SG-1原生质体经紫外线诱变并筛选链霉素抗性菌株，获得纳他霉素高产菌株。在上述高产突变株中选择4株作为亲本进行基因组重排育种，筛选得到了高产重排菌株，其中一株重排菌株S．gilvosporeus GS-74的纳他霉素产量为3574mg／L。为产量最高的亲本菌株的153％，比原始出发菌株SG-1提高1.17倍。     

Isolation and identification of 3-propylidene-delta 1-pyrroline-5-carboxylic acid, a biosynthetic precursor of lincomycin.

An accumulated lincomycin intermediate in UC 8292, a lincomycin nonproducing strain of Streptomyces lincolnensis, has been isolated and purified by employing an assay system based on complementation of UC 11066, another lincomycin nonproducing strain of S. lincolnensis. The structure of the purified intermediate is shown to be 3-propylidene-delta 1-pyrroline-5-carboxylic acid, or 1, 2, 3, 6-tetradehydro-propylproline by mass spectrometry and NMR spectroscopic studies. Based on the structure of this newly found intermediate, a biosynthetic pathway for propylproline is proposed as tyrosine-->L-3-hydroxytyrosine (Dopa)-->-->-->-->3-propylidene-delta 1-pyrroline-5-carboxylic acid-->3-propyl-delta 2-pyrroline-5-carboxylic acid-->propylproline.     

C-19393 S2 and H2, new carbapenem antibiotics. I. Taxonomy of the producing strain, fermentation and antibacterial properties

C-19393 S2 and H2 are new carbapenem antibiotics produced by a streptomycete. The producing strain was taxonomically studied and named Streptomyces griseus subsp. cryophilus. Cobaltous compounds were necessary for production of the antibiotics. C-19393 S2 and H2 showed a broad spectrum of antibacterial activities with C-19393 H2 being 8 approximately 120 times more active than C-19393 S2. They also exhibited beta-lactamase-inhibiting activities and acted synergistically with ampicillin and cefotiam against clinical isolates resistant to beta-lactam antibiotics.     

Frequent loss and restoration of antibiotic production by Streptomyces lasaliensis

Antibiotic nonproducing variants of Streptomyces lasaliensis NRRL 3382R, which makes the polyether antibiotic lasalocid A (Las) and the quinoxaline antibiotic echinomycin (Ech), arose at a frequency of 3-11% after treatment with three different mutagens or regeneration of protoplasts compared with a spontaneous frequency of less than 0.1%. Cosynthesis of lasalocid A was not observed upon testing a large number of Las- mutants in different pair-wise combinations, nor did these mutants accumulate probable intermediates of lasalocid A biosynthesis. These results suggest that loss of the las genes or their expression is induced at a high frequency by mutagenic treatments. In fusions of protoplasts of a strain with the las+ ech+ spo+ nic-1 rif-3 markers with strains bearing the Las- LasS Ech- Bld- (or spo+) str-1 markers, Las+ Ech+ Spo+ StrR progeny were produced at a 61-89% frequency compared with a 1-9% frequency of StrR antibiotic producing progeny with the nic-1 or rif-3 genotypes. The more frequent restoration of antibiotic production than prototrophy or rifampicin sensitivity indicates that these antibiotic characters did not behave as normal chromosomal markers. Therefore the genetic instability might be due to the involvement of a plasmid in antibiotic production. The apparent lack of infectious transfer of the Las+ character to Las- parents in conjugal matings between the few strains tested and no correlation between the presence of a large plasmid, pKSL, and lasalocid A production in several strains of S. lasaliensis do not favor the latter hypothesis, but they do not conclusively disprove it. Consequently, we suggest that a plasmid or another mobile genetic element is controlling antibiotic production in S. lasaliensis.     

原生质体诱变及高通量筛选选育链霉素高产菌株

目的 通过诱变和筛选方法的研究，提高灰色链霉菌(Streptomyces griseus)生产链霉素的水平。方法 优化灰色链霉 菌的原生质体的生成和再生条件，并对得到的原生质体进行紫外诱变，然后利用微孔板高通量方式对获得菌株进行筛选。结果 经过诱变选育获得一株菌NP-11703，其链霉素产量在100L罐上比出发菌株提高了21.8%。结论 用紫外诱变原生质体，可以有 效提高灰色链霉菌产链霉素的能力。结合高通量筛选模型的应用，可大大加快高产菌株的筛选效率。     

Mutagenesis of the rapamycin producer Streptomyces hygroscopicus FC904

Rapamycin (RPM) is produced by Streptomyces hygroscopicus FC904 isolated from soil in Fuzhou, China. It is a triene macrolide antibiotic with potential application as an immunosuppressant and drug for human gene therapy. In an attempt to improve rapamycin production, mutation and screening of the parent culture have been carried out. Thousands of survivors were obtained after mutagenesis by NTG (3 mg/ml) and UV (30 W, 15 cm, 30 seconds) of spore suspensions. None showed improved production of RPM. We determined the susceptibility to antibiotics of S. hygroscopicus FC904 by two fold dilutions of antibiotics in oatmeal agar plates. It was found that the strain was resistant to penicillin, erythromycin, RPM, tetracycline and chloramphenicol, but susceptible to mitomycin C (MIC, 10 microg/ml) and aminoglycosides such as gentamicin (MIC, 0.1 microg/ml), kanamycin (MIC, 0.1 microg/ml) and streptomycin (MIC, 0.3 microg/ml). Protoplasts of strain FC904 were prepared after finding the best conditions for their formation. They were treated with gentamicin, erythromycin, mitomycin C and NTG. Surprisingly, gentamicin was especially effective for obtaining higher RPM-producing mutants. Mutant C14 was selected by exposing the protoplasts of the parent strain FC904 to 1 microg/ml of gentamicin at 28 degrees C for 2 hours. A higher RPM-producing mutant (C14-1) was obtained from the protoplasts of mutant C14 treated with gentamicin, and its titer was 60% higher than that of the parent strain FC904 by HPLC analysis. Another improved mutant (C14-2) was obtained from the spores of mutant C 14 treated with 1 microg/ml of gentamicin plus 2 mg/ml of NTG at 28 degrees C for 2 hours. Mutant C14-2 had a titer 124% higher than FC904. The possible mechanism for the effect of gentamicin by using protoplasts or spore suspensions will be discussed, i.e. the possibility of gentamicin being a mutagen or a selective agent.