Intergeneric protoplast fusion between Fusobacterium varium and Enterococcus faecium for enhancing dehydrodivanillin degradation

Intergeneric protoplast fusion between Fusobacterium varium (Pcs Glu+) and Enterococcus faecium (Pcr Glu-) was performed under strictly anaerobic conditions to improve dehydrodivanillin (DDV) degradation. The fusion frequency obtained from the selective medium (Pc+ Glu-) was about 0.9 X 10(-5) to 1.3 X 10(-5). The seven fusants isolated were all gram-negative anaerobes with rod shapes like that of F. varium and with main phenotypical properties of cocci like those of E. faecium such as esculin and starch hydrolysis, milk clotting, and lactate production. Five fusants showed enhanced DDV degradation activities that were 2 to 4 times higher than those of parental strains. Genetic relatedness between a fusant (FE7) and the parents was estimated by DNA-DNA Southern blot hybridization with 32P-labeled chromosomal DNA fragments of F. varium and E. faecium as respective probes. The fusant FE7 presented a very high cross-hybridization with both probes, indicating a high DNA homology between the fusant and both parental strains. Almost all the fusants obtained here have stably kept the properties described above for about 2 years. These results suggest that intergeneric gene transfer takes place through protoplast fusion and that the fusants that were obtained are stable recombinants.     

Intergeneric protoplast fusion between Fusobacterium varium and Enterococcus faecium for enhancing dehydrodivanillin degradation.

Intergeneric protoplast fusion between Fusobacterium varium (Pcs Glu+) and Enterococcus faecium (Pcr Glu-) was performed under strictly anaerobic conditions to improve dehydrodivanillin (DDV) degradation. The fusion frequency obtained from the selective medium (Pc+ Glu-) was about 0.9 X 10(-5) to 1.3 X 10(-5). The seven fusants isolated were all gram-negative anaerobes with rod shapes like that of F. varium and with main phenotypical properties of cocci like those of E. faecium such as esculin and starch hydrolysis, milk clotting, and lactate production. Five fusants showed enhanced DDV degradation activities that were 2 to 4 times higher than those of parental strains. Genetic relatedness between a fusant (FE7) and the parents was estimated by DNA-DNA Southern blot hybridization with 32P-labeled chromosomal DNA fragments of F. varium and E. faecium as respective probes. The fusant FE7 presented a very high cross-hybridization with both probes, indicating a high DNA homology between the fusant and both parental strains. Almost all the fusants obtained here have stably kept the properties described above for about 2 years. These results suggest that intergeneric gene transfer takes place through protoplast fusion and that the fusants that were obtained are stable recombinants.     

单亲灭活柠檬酸杆菌与奇球菌原生质体融合

利用单亲灭活原生质体技术对柠檬酸杆菌和奇球菌原生质体进行了融合,考察了原生质体制备条件与融合中的影响因素。试验表明,随着溶菌酶浓度,酶解时间的增加和温度的上升,原生质体的形成率呈上升趋势,而再生率则逐渐下降。另外,正交试验结果表明,在PEG(6000)浓度40%、融合时间10min、融合温度42°C、pH值为8的条件下促融,最大融合频率可达2.74×10-7。筛选出来的融合子传代10次,性状稳定。进一步研究发现融合子在铀溶度为85mg/L时,比柠檬酸杆菌耐受性好;融合子和亲本吸附比较试验中,融合子总体吸附性能比柠檬酸杆菌略高。研究结果为耐辐射基因工程菌的构建提供了基础。     

Fusant Trichoderma HF9 with enhanced extracellular chitinase and protein content

Strain improvement was carried out to obtain higher chitinase and protein by inter-specific protoplast fusion between Trichoderma harzianum and Trichoderma viride. Fusant HF9 and parental strains of Trichoderma were compared for chitinase and protein production. 1% of glucose, sucrose and fungal cell wall (Rhizoctonia solani), were used as carbon source for cultivation of Trichoderma and fungal cell wall was the best to induce chitinase and protein. Usage of 0.5% colloidal chitin for the fungal growth under aerated conditions at pH 6.5 and 28°C led to higher chitinase and protein production. In these conditions fusant Trichoderma HF9 in comparison with parent strains had 3-, 2.5- and 1.5-fold increase of total chitinase, specific chitinase and protein, respectively. SDS-PAGE analysis revealed that it had 9 major protein bands with up-regulation compared to parent strains. Amino acid analysis showed that protein of culture filtrate of T. harzianum, T. viride and fusant Trichoderma HF9 had 8, 6 and 10 amino acids, respectively. The results obtained suggested that fusant HF9 could be an integration of T. harzianum and T. viride through protoplast fusion.     

克鲁维酵母与酿酒酵母属问原生质体融合构建高温酵母菌株*

通过PEG诱导碘乙酸灭活呼吸缺陷克鲁维酵母（Kluyveromycessp．Y034）原生质体与酿酒酵母（SaccharomycescerevisiaeA001）原生质体融合,获得45℃发酵产酒率高达8．7％的高温酵母菌株AY006。线粒体缺失和氯霉素抑制可显著降低高密度原生质体回复抑制效应。对融合子菌落形态、同工酶性质和高温发酵等方面分析,融合株表达了耐温和高产酒率双亲优良性状,证实其杂种特征。     

Regeneration and molecular characterization of an intergeneric hybrid between Graphium putredinis and Trichoderma harzianum by protoplasmic fusion

The fungal strains Graphium putredinis and Trichoderma harzianum were selected as parents for fusant development. Protoplasts were isolated using the combination of lysing enzymes Novozym 234 and cellulase with 0.6 M KCl as osmotic stabilizer. The optimum conditions for release of viable protoplasts from the fungal mycelium viz. age of the mycelium, lytic enzymes, osmotic stabilizers, pH, incubation period and regeneration medium were determined. Intergeneric protoplast fusion was carried out using 50% polyethylene glycol with calcium chloride (CaCl₂) and glycine buffer and the conditions for effective protoplast fusion, viz. fusogen, osmotic stabilizer, pH, incubation period and regeneration medium were optimized. At optimum conditions, the regeneration frequency of the fused protoplasts on potato dextrose agar (PDA) medium and fusion frequency were calculated. The regeneration frequency on non-selective (PDA) and selective media (PDA amended with starch) was determined for the parental and fusant strains in which, fusant showed a higher rate of regeneration. Fusant formation was confirmed by morphological markers (colony morphology and spore size and shape) and genetical markers like, mycelial protein pattern, restriction digestion pattern and random amplified polymorphic DNA (RAPD) analysis. The efficiency of these parental strains and their intergeneric fusant in the production of hydrolytic enzymes – amylases (treatment plant for sago factory effluent), cellulases (bioethanol), xylanases (bleaching agents for waste paper pulp) and proteases (additives in commercial detergents) – have probable applications in various industrial processes.     

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...     

原生质体融合技术构建棕榈油酸高产酵母菌株

采用原生质体融合技术进行产棕榈油酸酵母Saccharomy cescerevisiaeNo.12.926和产脂酵母RhodotorulaNo.12.908的融合研究,获得了棕榈油酸高产酵母工程菌株。实验结果表明,原生质体形成的最佳条件为:对数期酵母No.12.926和No.12.908用2%蜗牛酶于30℃分别酶解1.5和2h。在最佳条件下,酵母No.12.926和No.12.908原生质体形成率分别为94%和80%,再生率分别为75%和60%。原生质体融合由聚乙二醇诱导。将得到的融合子进行多次传代培养优选,获得了遗传性状稳定的融合菌株。融合子的生物量为亲株的两倍多,其细胞形态和菌落颜色与亲株有差别。产脂和产棕榈油酸分析表明,融合子的产脂量为菌体干重的48.53%,其中棕榈油酸占油脂总量的47.29%,为菌体干重的22.95%。     

以原生质体融合技术构建广谱抗噬菌体菌株

利用１５株不同品系的噬菌体作筛子筛选的噬菌体抗性自发突变株只对其相应的筛于噬菌体具抗性,为窄谱抗株。利用原生质体融合技术把北京棒杆菌７３３８与钝齿棒杆菌Ｔｓ－Ｌｉ进行融合得融合于ＺＧ８８,通过噬菌体吸附试验,血清学试验证明ＺＧ８８细胞表面结构发生了较大的改变,失去了噬菌体的吸附位点。因此ＺＧ８８是广谱稳定的噬菌体抗性菌株。     

Identification of protoplast fusion strain Fhhh by randomly amplified polymorphic DNA

A functional strain Fhhh was constructed through protoplast fusion of three parental strains (Phanerochaete chrysosporium, Saccharomyces cerevisiae and native bacterium YZ) to improve the degradation efficiency of purified terephthalic acid wastewater. Randomly amplified polymorphic DNA (RAPD) and scanning electron microscope (SEM) analysis were applied to identify and confirm the fusant Fhhh through phenotypic and genetic relationship. The result of SEM analysis demonstrated that the cell shape of fusant Fhhh differed from all three parental strains. RAPD analysis of 40 arbitrary primers generated a total of 1,135 bands. The genetic similarity indices between Fhhh and parental strains Phanerochaete chrysosporium (PC), Saccharomyces cerevisiae (SC) and native bacterium (YZ) were 34.01%, 33.16%, and 35.97%, respectively. The targeted-gene PCR results showed that Fhhh inherited the DNA fragments of mnp and lip genes from parental strain PC and FLO1 gene fragment from parental strain SC. Our results suggested protoplast fusion technique may be considered as a promising technique in environmental pollution control.     

原生质体融合子代的筛选和鉴定

介绍了绿色木霉N6和黑曲霉856原生质体融合子代的研究。经传代、发酵、筛选,从11株初筛的融合子中得到了3株纤维素酶活高且稳定的菌株AT23、AT16、AT34,其CMC酶活分别为亲本绿色木霉N6的2．2倍、1．4倍、1．2倍。并对其进行制霉菌素抗性试验和可溶性蛋白质凝胶电泳分析鉴定。试验结果证明了AT23、AT16、AT34是基因发生了重组的融合子且具有杂种优势。     

A fusant of Sphingomonas sp. GY2B and Pseudomonas sp. GP3A with high capacity of degrading phenanthrene

A fusant strain F14 with high biodegradation capability of phenanthrene was obtained by protoplast fusion between Sphingomonas sp. GY2B (GenBank DQ139343) and Pseudomonas sp. GP3A (GenBank EU233280). F14 was screened and identified from 39 random fusants by antibiotic tests, scanning electron microscope (SEM) and randomly amplified polymorphic DNA (RAPD). The result of SEM analysis demonstrated that the cell shape of fusant F14 different from parental strains. RAPD analysis of 5 primers generated a total of 70 bands. The genetic similarity indices between F14 and parental strains GY2B and GP3A were 27.9 and 34.6 %, respectively. F14 could rapidly degrade phenanthrene within 24 h, and the degradation efficiency was much better than GY2B and GP3A. GC–MS analysis of metabolites of phenanthrene degradation indicated F14 had a different degradation pathway from GY2B. Furthermore, the fusant strain F14 had a wider adaptation of temperatures (25–36 °C) and pH values (6.5–9.0) than GY2B. The present study indicated that fusant strain F14 could be an effective and environment-friendly bacterial strain for PAHs bioremediation.     

克鲁维酵母种间原生质体融合的研究

乳酸克鲁维酵母(Kluyueromyces lactis Y12—1)和脆壁克鲁维酵母(K.fragilis8554)是乳糖酶生产菌株。应用原生质体融合技术进行了两菌株种问融合的研究。通过试验．原生质体形成及再生的最佳条件为：对数期的细胞,2％的蜗牛酶．30℃酶解30分钟．原生质体形成率90％以上,再生率20%左右。原生质体融合由聚乙二醇(PEG)诱导。K.lactisY12-l不能旋酵菊糖;K.fragilis 8554不能同化D－松三糖和麦芽糖;利用二菌株自身的营养缺陷性质获得融合子。融合子既能发酵菊糖又能同化D－松三糖和麦芽糖;融合子的DNA含量约为二亲株之和;融合子的菌落形态与亲株相比有一定差别．在以乳糖为碳源的培养基中,融合子的乳糖酶产量提高14一l6％;连续15次传代,融合子稳定。     

芽孢杆菌和欧文氏菌的原生质体融合的研究

采用原生质体融合技术对带有链霉素标记的芽孢杆菌B12 13 2 和带有红霉素标记的欧文氏菌E2 6 2 3 进行原生质体融合。采用选择性及非选择性培养基对融合子进行 10次重复性传代试验后 ,获得稳定的融合子 2 84株 ,经重复性苎麻脱胶试验 ,从中获得 6株脱胶效果较好的融合子。     

基因组重排选育胸苷磷酸化酶高产菌株

以短乳杆菌为研究对象,通过基因组重排技术选育胸苷磷酸化酶高产菌株。首先采用紫外复合诱变筛选出EA42、EB27作为基因组重排育种的亲本并制备成原生质体,分别采用紫外照射50min和60℃水浴加热60min双亲灭活原生质体,然后用质量分数40％PEG6000,30℃恒温诱导融合10min进行基因组重排。经过3轮基因组重排育种,成功选育出3株胸苷磷酸化酶高产菌株,其中菌株F3-36在菌体发酵量提高的前提下,进行5次传代测试其胸苷磷酸化酶活均在2．500U／mg湿菌体,比原始菌株酶活提高了260％。     

Batch phenol degradation by candida tropicalis and its fusant

Phenol degradation by Candida tropicalis and its fusant, which is produced using protoplast fusion as a selective technique, is evaluated under batch and high concentration conditions. The respirometric data show that oxygen uptake activities of both yeast strains peak at pH 7.0 and 32 degrees C, but the fusant is more active than the control strain. Although the data show that both yeast strains are capable of sustaining discernible degradation in the presence of phenol inhibition, however, the C. tropicalis fusant is capable of attaining better phenol degradation than the control strain and it is less susceptible to phenol inhibition. Under the conditions tested, C. tropicalis is completely inhibited at phenol concentrations >/=3,300 mg/L, whereas for the C. tropicalis fusant complete inhibition is absent until phenol concentrations are >/=4, 000 mg/L. The observed cell yields of both yeast strains are virtually identical and remain fairly constant at approximately 0.5 mg MLVSS/mg C6H5OH (MLVSS: mixed liquor volatile suspended solids). Copyright 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 391-395, 1998.     

Amylase hyper-producing haploid recombinant strains of Thermomyces lanuginosus obtained by intraspecific protoplast fusion

Amylase hyper-producing, catabolite-repression-resistant, recombinant strains were produced by intraspecific protoplast fusion of thermophilic fungus Thermomyces lanuginosus strains, using well-characterized, morphological, and 2-deoxy-D-glucose resistant markers. The fusant heterokaryons exhibited enhanced amylase activities as compared to the amylase hyper-producing parental strain (T2). Diploids derived from heterokaryons segregated to stable haploid recombinant strains. In the haploid strain (Tlh 4q), approximately 5-fold higher specific activities of alpha-amylase and glucoamylase in the culture filtrate were observed as compared to the wild-type strain (W0).     

凤尾菇和盖囊侧耳原生质体非对称融合的研究

盖囊侧耳的双核体原生质体经过灭活处理作为供体与带有营养缺陷型标记的凤尾菇单核体原生质体受体融合,得到大量生长速度和菌落形态差异较大的融合子,这些融合子主要显示了供体菌株的特性,数次转接和进行原生质体再分离后,有的融合子再生菌株恢复了供体亲本的遗传特性,同时获得了一些新的生理特证。结果表明原生质体非对称融合可以作为食用菌原生质体融合育种的一种有效方式。     

双亲灭活制备粘质沙雷氏菌和红曲霉的跨界产色素融合子

目的:采用双亲灭活原生质体技术制备粘质沙雷氏菌和红曲霉的跨界产色素融合子,并测定其抑菌活性。方法:经0.2%溶菌酶处理获得粘质沙雷氏菌的原生质体并热灭活;经混合酶(0.8%溶菌酶+1.2%蜗牛酶+1.6%纤维素酶)处理获得红曲霉的原生质体并紫外灭活;用含25%PEG的原生质体融合剂进行促融合与再生。观察融合子的菌落形态和色素合成能力,测定融合子色素提取物对金黄色葡萄球菌的抑制活性。结果:在优化条件下,粘质沙雷氏菌原生质体的形成率为92.58%,红曲霉原生质体形成数约为106个/mL,两菌原生质体灭活率均为100%。共获得13个融合子,9个能产红色素,融合率为1×10-5%。其中8个融合子的95%乙醇提取物对金黄色葡萄球菌表现出不同程度的抑制。结论:采用双亲灭活原生质体技术,能够制备具有抑菌活性的粘质沙雷氏菌和红曲霉的跨界产色素融合子。     

双亲灭活制备粘质沙雷氏菌和红曲霉的跨界产色素融合子

目的：采用双亲灭活原生质体技术制备粘质沙雷氏菌和红曲霉的跨界产色素融合子,并测定其抑菌活性。方法：经0.2%溶菌酶处理获得粘质沙雷氏菌的原生质体并热灭活;经混合酶（0.8%溶菌酶＋1.2%蜗牛酶＋1.6%纤维素酶）处理获得红曲霉的原生质体并紫外灭活;用含25%PEG的原生质体融合剂进行促融合与再生。观察融合子的菌落形态和色素合成能力,测定融合子色素提取物对金黄色葡萄球菌的抑制活性。结果：在优化条件下,粘质沙雷氏菌原生质体的形成率为92.58%,红曲霉原生质体形成数约为106个/mL,两菌原生质体灭活率均为100%。共获得13个融合子,9个能产红色素,融合率为1×10-5%。其中8个融合子的95%乙醇提取物对金黄色葡萄球菌表现出不同程度的抑制。结论：采用双亲灭活原生质体技术,能够制备具有抑菌活性的粘质沙雷氏菌和红曲霉的跨界产色素融合子。