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％。     

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...     

用Genome shuffling技术选育紫杉醇高产菌株

以树状多节孢（Nodulisporium sylviforme）紫杉醇产生菌为研究对象,探索了紫杉醇产生菌的基因组重排育种的基本规律,重点研究了紫杉醇产生菌的原生质体融合和基因组重排育种的方法．采用薄层层析（TLC）、高效液相色谱（HPLC）和质谱（MS）分析筛选重组子,通过四轮基因组重排成功选育出了3株遗传稳定的高产紫杉醇菌株,其中一株重排菌株F4-26的发酵液中紫杉醇含量达到516-37μg几,比原始出发菌株NCEU-1紫杉醇产量提高了64．41％,比亲本菌株紫杉醇产量提高了31．52％-44．72％．     

基因组重排筛选高产谷氨酰胺转胺酶菌株

谷氨酰胺转胺酶(TGase)的产量不足的问题一直限制其工业化生产规模,故采用基因组重排的方法,筛选高产谷氨酰胺转胺酶菌株。通过对不同制备条件下原生质体纯度和形成率的考量,获得制备原生质体的最优条件为以6mg/ml的溶菌酶浓度进行酶解,酶解时间2h。再优化融合条件,以2min紫外灭活和40min热灭活结合的方法挑选出融合子。通过两轮基因组重排,经过96孔板发酵高通量筛选和摇瓶发酵复筛验证,获得了一株产酶达7.12U/ml的茂源链霉菌,相比最初选用菌株的平均酶活提高65.5%。发酵结果显示,酶活提高的原因可能是在重组后原酶成熟更快、更彻底,且得到的菌株遗传稳定性良好。证明基因组重排能够有效提高菌株的产酶水平,同时为谷氨酰胺转胺酶产量提高提供理论依据。     

基因组重排技术选育乙醇高产菌株

里氏木霉（Trichoderma reesei）被认为是最合适联合生物加工（consolidated bioprocessing）的微生物之一。原始里氏木霉菌株产乙醇能力太低,需要进一步提高其产酒量。我们通过基因组重排技术提高了里氏木霉菌株产乙醇能力和乙醇耐受力。首先对CICC40360菌株孢子进行NTG诱变得到正向突变菌株,再以此为出发菌株进行基因组重排。进行基因组重排后,重组菌株在含不同乙醇浓度的原生质体再生培养基上进行筛选。突变菌株和原始菌株一起做摇瓶发酵实验进行比较以确定产乙醇能力的提高。经过两轮基因组重排后,筛选获得表现最优异的重组菌S2-254。该菌株能在利用50g/l葡萄糖发酵出6.2g/l乙醇,同时能耐受3.5% （v/v）浓度乙醇。上述结果表明,本实验采用的基因组重排技术能够有效而且快速获得具有目的性状的优良菌株。     

基因组重排筛选高产谷氨酰胺转胺酶菌株

谷氨酰胺转胺酶(TGase)的产量不足的问题一直限制其工业化生产规模,故采用基因组重排的方法,筛选高产谷氨酰胺转胺酶菌株。通过对不同制备条件下原生质体纯度和形成率的考量,获得制备原生质体的最优条件为以6mg/ml的溶菌酶浓度进行酶解,酶解时间2h。再优化融合条件,以2min紫外灭活和40min热灭活结合的方法挑选出融合子。通过两轮基因组重排,经过96孔板发酵高通量筛选和摇瓶发酵复筛验证,获得了一株产酶达7.12U/ml的茂源链霉菌,相比最初选用菌株的平均酶活提高65.5%。发酵结果显示,酶活提高的原因可能是在重组后原酶成熟更快、更彻底,且得到的菌株遗传稳定性良好。证明基因组重排能够有效提高菌株的产酶水平,同时为谷氨酰胺转胺酶产量提高提供理论依据。     

用基因组重排技术选育赖氨酸高产菌株

摘要：【目的】以北京棒杆菌（Corynebacterium pekinense）1为研究对象,选育赖氨酸高产菌株,并探索赖氨酸产生菌基因组重排育种的基本规律。【方法】利用基因组重排技术选育赖氨酸高产菌株。【结果】通过四轮基因组重排成功选育出了5株遗传稳定的高产赖氨酸菌株,其中1株重排菌株赖氨酸产量达到16.95 g/dL,比原始菌株Corynebacterium pekinense 1赖氨酸产量提高了37.14%,比亲本菌株赖氨酸产量提高了17.46％~31.19%。【结论】首次采用基因组重排技术改良赖氨酸产生菌,成功选育出了5株产量较稳定的高产赖氨酸菌株,具有潜在的应用价值。     

菊粉酶产生菌的筛选及~(60)Co诱变选育

目的:从新疆石河子盐碱地菊芋生长根际土壤中分离筛选高产菊粉酶活力菌株。方法:通过稀释平板涂布法分离微生物;利用60Co诱变选育,96孔板筛选突变菌株;采用3,5-二硝基水杨酸比色法测定菊粉酶酶活。结果:分离到12株具有菊粉酶活力的菌株,复筛得到1株高产菊粉酶活力菌株,将其命名为G-60;以此菌株为出发菌株进行60Co诱变,利用96孔板对诱变菌株进行筛选,经摇瓶发酵酶活测定,得到1株高产菊粉酶酶活的突变株,酶活达46.62 U/mL,是未诱变菌株酶活的2.72倍。结论:经诱变得到1株高产菊粉酶活力的突变菌株。     

菊粉酶产生菌的筛选及60Co诱变选育简

目的：从新疆石河子盐碱地菊芋生长根际土壤中分离筛选高产菊粉酶活力菌株。方法：通过稀释平板涂布法分离微生物;利用^60Co诱变选育,96孔板筛选突变菌株;采用3,5-二硝基水杨酸比色法测定菊粉酶酶活。结果：分离到12株具有菊粉酶活力的菌株,复筛得到1株高产菊粉酶活力菌株,将其命名为G-60;以此菌株为出发菌株进行^60Co诱变,利用96孔板对诱变菌株进行筛选,经摇瓶发酵酶活测定,得到1株高产菊粉酶酶活的突变株,酶活达46．62U/mL,是未诱变菌株酶活的2．72倍。结论：经诱变得到1株高产菊粉酶活力的突变菌株。     

基因组改组技术快速提高扩展青霉碱性脂肪酶产量

应用基因组改组技术快速提高扩展青霉碱性脂肪酶的产量。采用经过多代诱变的碱性脂肪酶产生菌扩展青霉(Penicillium expansum)FS8486以及分离自新疆火焰山口土样的溜曲霉(Aspergillus tamarii)FS-132作为出发菌株,经过两轮基因组改组,得到数株优良子代。其中一株酶活较出发菌株FS8486提高317%。对亲本与子代菌株的形态型、RAPD(随机扩增多态性DNA)多态性和脂肪酸组成分析初步确定筛选获得的菌株为亲本的改组子代。首次将基因组改组技术成功应用于真核微生物基因组改造,短期内使目标代谢产物获得提高,这对于在真核微生物育种中进一步推广该技术具有重要意义。     

豆豉纤溶酶产生菌的筛选及诱变

从广泛收集的豆豉成品及半成品中筛选到数株菌落形态各异且具有纤溶酶活性的菌株.分别采用亚硝酸和紫外线对豆豉纤溶酶产生菌DC-12进行诱变育种,成功筛选到了3株突变株,其纤溶酶产量较出发菌株分别提高了3.6、3.7和4.75倍.     

枯草芽胞杆菌基因组混组方法

基因组混组作为一种育种方法,通过循环原生质体融合等手段,使得不同菌株来源的基因组能够得到充分重组,增加将正向突变整合到同一重组子中的机会。使用4株带有4种不同标记的枯草芽胞杆菌亲本为初始菌株,通过循环转化、循环转导或循环原生质体融合的手段进行基因组混组,统计后代中非亲本类型占整个群体的比例,以衡量基因组混组的效果。分别经过5轮循环原生质体融合、循环转化或者循环转导,结果显示,重组程度较高者在后代群体中的比例较低,带有4种标记的后代未出现,带有3种标记的后代最高分别为4.53×10?4、1.64×10?4、4.47×10?3,明显低于文献报道的天蓝色链霉菌中同样实验的结果:带4种和3种标记的后代分别占2.5%、17%。对比上述实验的结果和文献报道的天蓝色链霉菌、乳杆菌基因组混组的结果,并结合计算机模拟循环融合过程,分析后认为:要达到较充分的基因组混组,需要有能够实现微生物细胞间高频重组的操作技术作为基础,重组频率应该不低于10?3～10?2数量级。     

Evolution of Streptomyces pristinaespiralis for resistance and production of pristinamycin by genome shuffling

Improvement of pristinamycin production by Streptomyces pristinaespiralis was performed by using recursive protoplast fusion and selection for improved resistance to the product antibiotic in a genome shuffling format. A 100-mug/ml pristinamycin resistant recombinant, G 4-17, was obtained after four rounds of protoplast fusion, and its production of pristinamycin reached 0.89 g/l, which was increased by 89.4% and 145.9% in comparison with that of the highest parent strain M-156 and the original strain CGMCC 0957, respectively. The subculture experiments indicated that the hereditary character of high producing S. pristinaespiralis G 4-17 was stable. It is concluded that genome shuffling improves the production of pristinamycin by enhancing product-resistance in a stepwise manner. Pristinamycin fermentation experiments by recombinant G 4-17 were carried out in a 5-l fermentor, and its production of pristinamycin reached 0.90 g/l after 60 h of fermentation.     

Screening and breeding of high taxol producing fungi by genome shuffling

To apply the fundamental principles of genome shuffling in breeding of taxol-producing fungi, Nodulisporium sylviform was used as starting strain in this work. The procedures of protoplast fusion and genome shuffling were studied. Three hereditarily stable strains with high taxol production were obtained by four cycles of genome shuffling. The qualitative and quantitative analysis of taxol produced was confirmed using thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and LC-MS. A high taxol producing fungus, Nodulisporium sylviform F4-26, was obtained, which produced 516.37 μg/L taxol. This value is 64.41% higher than that of the starting strain NCEU-1 and 31.52%–44.72% higher than that of the parent strains.     

Genome shuffling: Progress and applications for phenotype improvement

Although rational method and global technique have been successfully applied in strain improvement respectively, the demand for engineering complex phenotypes required combinatorial approach. The technology of genome shuffling has been presented as a novel whole genome engineering approach for the rapid improvement of cellular phenotypes. This approach using recursive protoplast fusion with multi-parental strains offers the advantage of recombination throughout the entire genome without the necessity for genome sequence data or network information. Genome shuffling has been demonstrated as an effective method, which is not only for producing improved strain but also for providing information on complex phenotype. In this review we attempt to present the advantage of genome shuffling, introduce the procedure of this technology, summarize the applications of this approach for phenotype improvement and then give perspective on the development of this method in the future.     

Genome-shuffling improved acid tolerance and L-lactic acid volumetric productivity in Lactobacillus rhamnosus

Genome shuffling is an efficient approach for the rapid improvement of industrially important microbial phenotypes. Here we improved the acid tolerance and volumetric productivity of an industrial strain Lactobacillus rhamnosus ATCC 11443 by genome shuffling. Five strains with subtle improvements in pH tolerance and volumetric productivity were obtained from the populations generated by ultraviolet irradiation and nitrosoguanidine mutagenesis, and then they were subjected for recursive protoplast fusion. A library that was more likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both ultraviolet irradiation and heat treatments. After three rounds of genome shuffling, four strains that could grow at pH 3.6 were obtained. We observed 3.1- and 2.6-fold increases in lactic acid production and cell growth of the best performing at pH 3.8, respectively. The maximum volumetric productivity was 5.77+/-0.05 g/lh when fermented with 10% glucose under neutralizing condition with CaCO(3), which was 26.5+/-1.5% higher than the wild type.     

Strain improvement of Sporolactobacillus inulinus ATCC 15538 for acid tolerance and production of D-lactic acid by genome shuffling

Improvement of acid tolerance and production of D-lactic acid by Sporolactobacillus inulinus ATCC 15538 was performed by using recursive protoplast fusion in a genome shuffling format. The starting population was generated by ultraviolet irradiation, diethyl sulfate mutagenesis, and pH-gradient filter and then, subjected for the recursive protoplast fusion. The concentration of lysozyme, time, and temperature for enzyme treatment were optimized by response surface methodology based on the central composite design. Based on contour plots and variance analysis, the model predicted a maximum Y (multiply protoplasts formation ratio by protoplasts regeneration ratio), 60.4%, and the corresponding above used values were 7.75 mg/ml lysozyme, 1.59 h, and 38°C. A pH-5-resistant recombinant, F3-4, was obtained after three rounds of genome shuffling and its production of D-lactic acid reached 93.4 g/l in a 5 L bioreactor, which was increased by 39.8% and 119% in comparison with that of UV generated strain and the original strain S. inulinus ATCC 15538, respectively. The subculture experiments indicated that F3-4 was genetically stable.     

基因组重排技术在开发新代谢产物中的应用

基因组重排是一种基于原生质体融合,并对原生质进行递推式融合的新型技术。随着基因组重排技术的不断发展和成熟,通过基因组重排获得新代谢产物的例子不断出现,表明该项技术作为新代谢产物开发的途径具有一定的应用前景。在此列举了基因组重排在开发新代谢产物方面的成果,包括基因组重排激活沉默基因产生新代谢产物;基因组重排引入单酶基因产生新抗生素;基因组重排互换基因模块产生杂合抗生素和基因组重排替换前体基因产生新抗生素的例子,并展望了其发展的趋势。     

Genome shuffling of Streptomyces viridochromogenes for improved production of avilamycin

Avilamycin is one of EU-approved antimicrobial agents in feed industry to inhibit the growth of multidrug-resistant Gram-positive bacteria. Here, we applied a process of combining ribosome engineering and genome shuffling to achieve rapid improvement of avilamycin production in Streptomyces viridochromogenes AS 4.126. The starting mutant population was generated by ⁶⁰Co γ-irradiation treatments of the spores. After five rounds of protoplast fusion with streptomycin-resistance screening, an improved recombinant E-219 was obtained and its yield of avilamycin reached 1.4 g/L, which was increased by 4.85-fold and 36.8-fold in comparison with that of the shuffling starter Co γ-316 and the ancestor AS 4.126. Furthermore, the mechanism for the improvement of shuffled strains was investigated. Recombinants with enhanced streptomycin resistance exhibited significantly higher avilamycin production and product resistance, probably due to the mutations in the ribosome protein S12. The morphological difference between the parent mutant and shuffled recombinant was observed in conidiospore, and hyphae pellets. The presence of genetic diversity among shuffled populations with varied avilamycin productivity was confirmed by randomly amplified polymorphic DNA analysis. In summary, our results demonstrated that genome shuffling combined with ribosome engineering was a powerful approach for molecular breeding of high-yield industrial strains.