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％。     

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.     

1,3-丙二醇产生茵基因组重排育种

[目的]本文以肺炎克雷伯氏杆菌为研究对象,利用基因组重排技术,提高其对发酵体系中主要产物的耐受性,获得1,3-丙二醇高产菌.[方法]以含预处理后的出发菌株的补料发酵终点液的96孔板为筛选方法,利用基因组重排技术育种.[结果]筛选到的5株高产菌株(LSG1,LSG2,LSG4,LSG5,LSG6),在3升罐批次发酵中的1...     

基因组重排技术在酵母菌育种中的应用

基因组重排(genome shuffling)技术是在传统诱变育种的基础上与细胞原生质体融合技术相结合一种新兴微生物菌种改良手段,由于该技术高效的正向突变效率和频率,近年来被广泛应用于酵母菌种的选育和改良。本文主要对基因组重排技术在酵母菌育种中的应用进行了综述。     

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。     

真菌紫杉醇/烷研究近况

紫杉醇（Taxol?）是天然抗癌药物,已在临床上广泛使用,市场需求量大,由于生产原料的制约,供需之间存在巨大缺口,价格仍然昂贵。利用真菌发酵生产紫杉醇是解决药源的一条新途径。对产紫杉醇内生真菌的多样性,紫杉醇真菌的采集鉴定、真菌紫杉醇提取和测定的一些经验,紫杉醇真菌分子生物及生物合成代谢研究进展等进行了综述。     

产紫杉醇菌株原生质体诱变育种的研究

对紫杉醇产生菌NCEU-1的原生质体进行了紫外线和氯化锂复合诱变,筛选制霉菌素抗性突变株,共筛选出了4株正突变株。经发酵筛选试验,获得了一株遗传性状稳定、高产紫杉醇的原生质体诱变菌株——UL_04-5,其紫杉醇产量从出发菌株的314.07μg/L提高至418.24μg/L。     

内生真菌产紫杉醇研究的回顾与展望

紫杉醇是一种高效、低毒、广谱的天然抗癌药物,是人类未来20年间最有效的抗癌药物之一。近年来的研究发现,从植物内生真菌中发酵生产紫杉醇被证明是解决药源问题的有效途径。本文概述了微生物发酵生产紫杉醇的研究进展,包括产紫杉醇内生真菌的分离、多样性和真菌产紫杉醇的优势,紫杉醇的提取和检测技术,同时对如何提高内生真菌紫杉醇产量进行了比较全面的综述。随着现代分子生物学技术和微生物发酵工程的发展,工业上大规模发酵生产紫杉醇将有望实现。     

Molecular breeding of genes, pathways and genomes by DNA shuffling

Existing methods for optimization of sequences by random mutagenesis generate libraries with a small number of mostly deleterious mutations, resulting in libraries containing a large fraction of non-functional clones that explore only a small part of squence space. Large numbers of clones need to be screened to find the rare mutants with improvements. Library display formats are useful to screen very large libraries but impose screening limitations that limit the value of this approach for most commercial applications. By contrast, in both classical breeding and in DNA shuffling, natural diversity is permutated by homologous recombination, generating libraries of very high quality, from which improved clones can be identified with a small number of complex screens. Given that this small number of screens can be performed under the conditions of actual use of the product, commercially relevant improvements can be reliably obtained.     

内生真菌紫杉醇生物合成的研究现状与展望

紫杉醇是重要的抗癌药物之一,已经证明其对多种癌症具有显著疗效。目前,人们主要是从红豆杉的树皮中提取、分离和纯化紫杉醇,但由于红豆杉为生长缓慢、散生、濒危的珍稀植物,且随着紫杉醇临床用途的不断拓宽,市场需求的稳定增长,单纯依靠从红豆杉树皮中提取紫杉醇已经无法满足日益增长的市场需求。为了解决紫杉醇的药源不足,科学家已把目光从红豆杉树分离提取紫杉醇转向了其他替代方法,如化学全合成、半合成、组织培养与细胞培养、微生物发酵法生产紫杉醇等。因此,了解内生真菌紫杉醇生物合成的分子基础和遗传调控机制,对解析内生真菌紫杉醇生物合成机制、构建高产紫杉醇基因工程菌株和早日实现内生真菌紫杉醇工业化生产具有重要的科学意义和现实意义。结合本课题组多年来的科研工作,概述了红豆杉细胞紫杉醇生物合成途径、内生真菌发酵生产紫杉醇的优势、产紫杉醇内生菌的分离研究现状和生物多样性及紫杉醇生物合成相关基因的研究现状。内生真菌生物发酵合成紫杉醇是可以无限生产、大量获取紫杉醇、解决紫杉醇药源短缺问题的很有前景的方法之一。     

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。     

全基因组重排育种技术提高产豆豉纤溶酶菌产酶量

枯草芽孢杆菌DC-12 是从豆豉里面筛选出来的,具有纤溶酶活性的菌株。本论文采用了全基因组重排技术提高DC－12的产酶量,首先通过对DC－12进行紫外诱变和亚硝酸诱变构建重组突变库,在研究其原生质体制备和再生的基础上,以其中4株诱变菌株作为直接亲本,采用电融合的方法进行两次多亲本的全基因组重排,结合双灭活的筛选方法,共筛选出2株酶活大大提高并能稳定遗传的菌株,使亲本菌株的酶活提高了4~5倍,最高达2710 IU/ml。     

微生物菌种选育中的基因组改组技术及其应用进展

该文论述了基因组改组技术的产生和原理、方法和特点,以及该技术的应用、意义及其发展前景.基因组改组技术是首先对微生物菌株进行诱变,筛选出正向突变的菌株,然后通过原生质体"递推式融合"使这些正向突变的若干个菌株进行基因组重组,从中筛选出符合育种要求的重组子,从而在短时间内获得性状得到大幅度提高的菌株.     

高产紫杉醇菌株的诱变选育及其差异表达基因消减cDNA文库的构建

[目的]选育高产紫杉醇菌株,并构建选育到的高产紫杉醇菌株与出发菌株HD1-3差异表达的cDNA消减文库.[方法]分别采用硫酸二乙酯和紫外线与硫酸二乙酯复合诱变处理菌株HD1-3孢子;以选育到的高产紫杉醇菌株为tester,菌株HD1-3为driver,应用抑制性消减杂交技术构建选育到的高产紫杉醇菌株与菌株HD1-3差异表达的cDNA消减文库.[结果]试验确定的Nodulisporium sylviforme紫杉醇产生菌HD1-3孢子复合诱变的适宜条件为:将106cfu/mL孢子悬液经过8%硫酸二乙酯处理15 min后,在电磁搅拌下,用紫外灯(30 w,距离30 cm)照射处理45 s,获得了1株遗传性状稳定、高产紫杉醇的突变株--UD14-11,其紫杉醇产量从出发菌株HD1-3的232.73±4.61μg/L提高至312.81±7.51μg/L;构建的文库滴度为1.2×107cfu/mL,阳性克隆率75.3%,片段大小主要集中在300 bp-1.0kb.[结论]选育到了1株遗传性状稳定、高产紫杉醇突变株;成功地构建了高产紫杉醇菌株UD14-11与菌株HD1-3差异表达的cDNA消减文库,为寻找、分离微生物生物合成紫杉醇相关基因和利用基因工程或代谢工程手段定向设计改造菌株奠定基础.     

Improved production of propionic acid using genome shuffling

Traditionally derived from fossil fuels, biological production of propionic acid has recently gained interest. Propionibacterium species produce propionic acid as their main fermentation product. Production of other organic acids reduces propionic acid yield and productivity, pointing to by‐products gene‐knockout strategies as a logical solution to increase yield. However, removing by‐product formation has seen limited success due to our inability to genetically engineer the best producing strains (i.e. Propionibacterium acidipropionici). To overcome this limitation, random mutagenesis continues to be the best path towards improving strains for biological propionic acid production. Recent advances in next generation sequencing opened new avenues to understand improved strains. In this work, we use genome shuffling on two wild type strains to generate a better propionic acid producing strain. Using next generation sequencing, we mapped the genomic changes leading to the improved phenotype. The best strain produced 25% more propionic acid than the wild type strain. Sequencing of the strains showed that genomic changes were restricted to single point mutations and gene duplications in well‐conserved regions in the genomes. Such results confirm the involvement of gene conversion in genome shuffling as opposed to long genomic insertions.     

Separation of a tungus producing taxol

An endogenous filariform fungus has been separated from a treenamed Taxus growing in the Aba region, Sichuan, China. The fungus is fermented in fluid medium for 3 weeks at 25℃, then the HPLC and MALDI-TOF analysis of the zymotic fluid show that the zymotic fluid contains taxol. So it is a fungus which can produce taxol. It is named Taxomyces sp. temporarily.     

Separation of a tungus producing taxol

An endogenous filariform fungus has been separated from a tree named Taxus growing in the Aba region, Sichuan, China. The fungus is fermented in fluid medium for 3 weeks at 25℃, then the HPLC and MALDI-TOF analysis of the zymotic fluid show that the zymotic fluid contains taxol. So it is a fungus which can produce taxol. It is named Taxomyces sp. temporarily.     

内生真菌发酵生产紫杉醇的研究现状与展望

抗癌药物紫杉醇已在临床上广泛应用,但受原料红豆杉树木短缺的制约,存在巨大的供需差距,而内生真菌发酵生产紫杉醇是解决紫杉醇药源问题的很有前景的途径之一.结合课题组多年来开展的科学试验研究工作,概述了内生真菌发酵生产紫杉醇的优势、产紫杉醇内生真菌的分离研究现状和生物多样性及提高内生真菌生物合成紫杉醇量的途径.     

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.     

紫杉醇产生菌及其工程菌株之间的AFLP遗传差异分析

采用AFLP技术对三株紫杉醇产生菌(HQD33、HQD43、HQD54)及HQD33菌株经复合诱变后再经原生质体诱变得到的两株诱变株UL50-6和UV40-19及其融合菌株J1-3进行了分析,结果表明三株紫杉醇产生菌分属不同的种属,同时首次从分子水平上证实Z35-8,J1-3为两株不同的融合子,为下一步的基因工程育种工作中的基因定位和分子克隆提供丰富可靠的分子标记。