紫外-激光复合诱变选育天冬氨酸转氨酶高产菌株

对天冬氨酸转氨酶产生菌大肠杆菌XJ-1原生质体进行紫外-激光复合诱变筛选,结果表明,复合诱变对该菌的原生质体有明显的致死作用。以致死率和正突变率为指标,确定了紫外和He-Ne激光照射的最佳时间分别为45 s和40min。在此条件下对大肠杆菌原生质体进行紫外-激光复合诱变,得到3株高产菌株,分别命名为XJ-1-45、XJ-1-86和XJ-1-99,酶活较出发菌株XJ-1分别提高了12.82%、17.37%和26.27%。传代培养表明突变株生产性能稳定。     

脱落酸高产菌的激光诱变效应研究

本实验对比紫外、 Ｈｅ Ｎｅ 激光、 Ｎｄ： Ｙ Ａ Ｇ 倍频脉冲激光对脱落酸产生菌的诱变效果。紫外诱变正变率为 ３８４％ ,负变率 ７６９％ ; Ｈｅ Ｎｅ 激光诱变正变率为 ５６％ ; Ｎｄ： Ｙ Ａ Ｇ 倍频脉冲激光诱变正变率为１９６％ 。 Ｎｄ： Ｙ Ａ Ｇ 倍频脉冲激光诱变辐照次数在 ４００ 次时效果较好,所得高产株效价提高率可达 ６０％ 以上     

激光复合诱变选育叶酸高产菌

利用高效液相色谱测定发酵液中叶酸含量,比较产朊假丝酵母(Candida utilis)、异常汉逊酵母(ftan-senula anomala)和枯草芽孢杆菌(Bacillus subtilis)产叶酸能力的高低,从而确定生产叶酸的最佳菌种.出发菌株经紫外照射诱变后,再采用激光复合诱变方式进行进一步的筛选,并对其传代稳定性进行研究,以期进一步获得稳产高产叶酸产生菌突变株.结果表明产朊假丝酵母产叶酸量最高.紫外照射3 min得到的Y1.4菌株产叶酸量与原始菌株相比,产量提高了33.8%.激光一紫外复合诱变后筛选出4株产量较高的菌株,其中以Y2.12产量最高.Y2.12产叶酸量与原始菌株相比,提高了65.8%.经传代培养分析,Y2.12诱变株的产量稳定.该结果表明,激光复合诱变是获得高产叶酸的有效途径.     

阿魏菇多糖高产菌的离子束和激光复合诱变育种

目的:诱变筛选阿魏菇多糖高产菌,探索食用菌的离子束和激光复合诱变育种方法。方法:尝试用阿魏菇菌丝单细胞为靶材,以离子束注入和激光辐照为复合诱变手段,采用营养缺陷型筛选办法定性初筛,摇瓶发酵定量复筛。结果:通过离子束注入诱变,获得了2株菌丝体多糖产量分别达到551.80mg/L和659.46mg/L、较1号出发菌株提高了46.5%和75.2%的多糖高产菌PFPH-1和PFPH-2;在此基础上,以激光辐照为复合诱变手段,获得了1株菌丝体多糖产量达到762.50 mg/L、较出发菌株PFPH-2提高了15.63%的多糖高产菌PFPH-3。结论:离子束和激光复合诱变育种方法在改良阿魏菇多糖高产性状方面诱变功效显著。     

常压室温等离子体-紫外复合诱变选育β-法尼烯高产菌株

【背景】大肠杆菌(Escherichia coli)由于生长性能优良、遗传背景清楚、遗传操作手段成熟,是合成β-法尼烯的合适生产菌,但其合成β-法尼烯的产量目前仍不能满足工业化生产的需求。【目的】通过诱变筛选技术选育β-法尼烯高产突变株。【方法】采用常压室温等离子体(atmosphericand room temperature plasma,ARTP)诱变技术和紫外线照射对出发菌株大肠杆菌EC-16进行复合诱变,并以异戊烯焦磷酸耐受性为选择压力进行平板初筛,之后进行摇瓶复筛,最后进行发酵罐验证。通过连续多代培养筛选到的高产突变菌株,观察其遗传稳定性。【结果】经复合诱变选育筛选出一株β-法尼烯高产突变株E.coliHVK-9,其产量高达22.1g/L,相比出发菌株提高了168.74%。【结论】采用ARTP-紫外复合诱变,再结合异戊烯焦磷酸抗性筛选的集成方法,使得诱变菌株的正突变率大大提高,可以有效地提高诱变菌株的β-法尼烯产量。突变株HVK-9作为工业化发酵生产菌种具有较好的遗传稳定性,为β-法尼烯的工业化生产和应用奠定了良好的基础。     

高产洛蒙真菌素洛蒙德链霉菌高通量筛选方法的建立与复合育种

[背景]洛蒙德链霉菌S015能生物合成具有广谱抗菌活性的吩嗪类化合物洛蒙真菌素。[目的]因S015菌株的洛蒙真菌素产量较低,将S015菌株经复合诱变育种和基因工程改造,提高洛蒙真菌素产量。[方法]建立洛蒙真菌素产生菌的高通量筛选方法,对出发菌株S0 15进行常压室温等离子体(atmospheric and room temperature plasma,ARTP)技术和紫外复合诱变,筛选得到高产菌株;并在高产菌株上敲除洛蒙真菌素的前体分支酸竟争途径中的关键基因trpE1、trpE2,再过表达全局调控基因afsR。[结果]利用洛蒙真菌素在紫外波长375 nm处的特征吸收峰,以及洛蒙真菌素浓度和375 nm处吸光度值的正相关关系,建立了基于24孔深孔板发酵和酶标仪快速检测的高通量筛选方法。经过6轮ARTP和紫外复合诱变及高通量筛选,从4 320株突变株中筛选得到遗传稳定的高产菌株M6,其洛蒙真菌素的产量为61.33 mg/L,是S015菌株的7.35倍;M6菌株的分支途径基因trpE1、trpE2双敲株的洛蒙真菌素产量为81.89 mg/L,是S015菌株的9.82倍;在该基因工程菌株中过表达全局调控基因afsR,产量为109.53 mg/L,是S015菌株的13.13倍。[结论]建立的高通量筛选方法可以有效筛选高产洛蒙真菌素的突变株,并且操作简单快速。通过ARTP和紫外复合诱变,结合高产株M6的基因工程改造,能进一步提升洛蒙真菌素的产量。     

激光诱变筛选高产植酸酶黑曲霉菌株的研究

用1.06um,15KHz高重复率声光调QNd：YAG激光以20W和15W的辐照功率分别照射产植酸酶的黑曲霉孢子液和原生质体液不同时间,检定再生菌落数并经透明圈初筛和摇瓶复筛,测定诱变菌株所产植酸酶酶活。结果表明：20W的功率辐照黑曲霉孢子1′以上,致死率近乎100…,15W功率辐照原生质体液20″-4′,存活率在8.571％-117.14％之间,正变率在27.45％-100％之间,正变辐度60.69％-124.96％,说明原生质体比孢子耐受激光诱变的能力强,并且诱变效果好。筛选到了一株酶活提高达3.75倍的单个变异菌株。     

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。     

产生鬼臼毒素菌株的诱变选育

目的:提高原始菌株鬼臼毒素的产量,以期组织工业化生产,从而扩大鬼臼类化合物的资源。方法:以交链孢霉Ty为研究对象,进行紫外线和He-Ne激光复合诱变,再对所获得高产菌株进行发酵条件的优化。结果:筛选到一株鬼臼毒素高产菌株Ty4-16-13,产量达4.213μg/L,比原始菌株提高96%。结论:诱变后的菌株经7次传代,产量较稳定,可用于工业化生产。     

曲酸产生菌激光诱变效应的研究

采用经紫外线(UV)、^60Co、亚硝基胍(NTG)复合诱变得到的黄曲霉曲酸产生菌(UCN7—12),进行激光诱变处理。研究证实在经过UV、^60Co、NTG诱变处理后,黄曲霉突变株用He—Ne激光与YAG激光进行诱变处理仍能提高产酸率,其中He—Ne激光辐照处理20min,正变率为12．1％,产量提高约13％。YAG激光辐照处理300sec,正变率16．7％,产量提高18．3％。说明上述两种激光对黄曲霉曲酸产生菌有一定的诱变效应。     

复合诱变法选育香兰素高转化率菌株

通过紫外线照射和He-Ne激光修复对香兰素生产菌链霉菌Streptomyces sp.L1936进行复合诱变,得到香兰素高产菌株Streptomyces sp.L1936-8。结果表明:出发菌株经过诱变处理后,脱乙酰酶酶活提高了75%,香兰素氧化酶酶活降低了37.5%,香兰素产量提高了33%。     

The effect of reducing light-harvesting pigment on marine microalgal productivity

The productivity was evaluated of a strain of Chlamydomonas perigranulata isolated from the RedSea. A mutant with small light-harvesting pigments(LHC-1) was obtained by UV mutagenesis. Thechlorophylls content of the wild type was twice ashigh as that of LHC-1, and the initial slope of thephotosynthesis-irradiance curve was higher in the wildtype. However, the maximum photosynthetic activity ona per cell basis was almost the same. It isconcluded that LHC-1 is a mutant with lesslight-harvesting pigment (LHP) than the wild type. Aspreviously reported, the mutant with lower LHP contenthas a higher productivity in a continuous culturesystem, so we compared the productivity of the wildtype and the mutant. The maximum productivity of LHC-1was 1.5 times higher than that of the wild type. Itis suggested that the technique of reducing thecontent of light-harvesting pigment should be madeavailable for other organisms.     

Identification and iterative combinatorial mutagenesis of a new naringinase-producing strain,Aspergillus tubingensis MN589840

Naringinase was mainly obtained by microbial fermentation, and mutagenesis was a major way for obtaining excellent mutants. The aim of this study was to screen out a high naringinase yielding mutant to enhance the potential application value of its industrialization and compare the effects of different mutagenic methods on the enzyme activity of the strain. A novel producing naringinase strain, Aspergillus tubingensis MN589840, was isolated from mildewed pomelo peel, later subjected to mutagenesis including UV, ARTP and UV-ARTP. After five rounds iterative mutagenesis, the mutants U1, A6 and UA13 were screened out with 1448·49, 1848·71, 2475·16 U mg⁻¹ enzyme activity, the naringinase productivity raised by 79·08, 123·56 and 206%, respectively. In addition, the naringinase activity of three mutants rose after each round of iterative mutagenesis. These results indicated that the mutagenesis efficiency of UV-ARTP was higher than that of single ARTP, and both are better than UV. In summary, the iterative UV-ARTP mutagenesis is an effective strategy for screening high naringinase-producing strains.     

ENHANCED β-GALACTOSIDASE PRODUCTION OF Aspergillus oryzae MUTATED BY UV AND LiCl

In order to breed a high-yield β-galactosidase-producing strain, Aspergillus oryzae was used as the parent strain and mutagenized with ultraviolet (UV) and UV plus lithium chloride (LiCl), respectively. After being mutagenized by UV, the β-galactosidase activity of mutant UV-15-20 reached 114.08 U/mL, which revealed a 49.22% increase compared with the original strain. A mutant UV-LiCl-38 with high β-galactosidase activity (121.42U/mL) was obtained after compound mutagenesis of UV and LiCl; the β-galactosidase activity of this mutant was 58.82% higher than that of the parent strain. Subculture testing indicated that UV-15-20 and UV-LiCl-38 had good hereditary stability and may be ideal strains for the production of β-galactosidase. Additionally, it was demonstrated that compound mutagenesis with UV and LiCl is an effective mutation method for breeding industrially interesting strains.     

耐高糖高产2,3-丁二醇产酸克雷伯氏杆菌的选育

以产酸克雷伯氏杆菌(Klebsiella oxytoca) ME-UD-3为出发菌株,经紫外线及硫酸二乙酯复合诱变后分别在葡萄糖浓度逐渐提高的液体培养基中进行富集培养,筛选获得了一株耐高糖的2,3-丁二醇高产突变菌株K. oxytoca ME-UD-3-4;该菌株的初始葡萄糖耐受浓度从出发菌株的120g/L提高到300g/L以上,在初始葡萄糖浓度为95 g/L的条件下发酵培养,与出发菌株相比发酵时间缩短了8h,2,3-丁二醇的产量由原来的38.5g/L提高到43.0g/L,生产强度从0.80 g/L·h提高到1.08 g/L·h,转化率达到了理论值的91%。     

耐温性L-谷氨酸发酵菌种的选育

应用基因组改组技术提高,L-谷氨酸生产菌在高温发酵条件下的谷氨酸产量。以天津短杆菌T6—13变异株SW07-1为原始亲株,分别经紫外线（UV）-硫酸二乙酯（DES）和X射线诱变,获得5株耐温性能略有提高的突变菌株。经2轮基因组改组,获得耐高温（能在44℃生长）的L-谷氨酸菌株F2-50。F2—50在38℃下,摇瓶发酵40h,发酵液中L-谷氨酸浓度比原始出发菌株提高了近41％,在41℃高温下,摇瓶发酵40h,L-谷氨酸浓度比原始出发菌株提高了近2倍。     

曲酸生产菌的诱变选育

Aspergillusoryzae2336经两次紫外诱变后筛选出突变菌株UV21012-1。该菌株曲酸产量比出发菌株提高了1．68倍;遗传性状稳定,传代5次曲酸产量基本不下降,而且发酵周期也比出发菌株明显缩短。     

Combining classical, genetic, and process strategies for improved precursor-directed production of 6-deoxyerythronolide B analogues

A process for the production of erythromycin aglycone analogues has been developed by combining classical strain mutagenesis techniques with modern recombinant DNA methods and traditional process improvement strategies. A Streptomyces coelicolor strain expressing the heterologous 6-deoxyerythronolide B (6-dEB) synthase (DEBS) for the production of erythromycin aglycones was subjected to random mutagenesis and selection. Several strains exhibiting 2-fold higher productivities and reaching >3 g/L total macrolide aglycones were developed. These mutagenized strains were cured of the plasmid carrying the DEBS genes and a KS1 degrees mutant DEBS operon was introduced for the production of novel analogues when supplemented with a synthetic diketide precursor. The strains expressing the mutant DEBS were screened for improved 15-methyl-6-dEB production, and the best clone, strain B9, was found to be 50% more productive as compared to the parent host strain used for 15-methyl-6-dEB production. Strain B9 was evaluated in 5-L fermenters to confirm productivity in a scalable process. Although peak titers of 0.85 g/L 15-methyl-6-dEB by strain B9 confirmed improved productivity, it was hypothesized that the low solubility of 15-methyl-6-dEB limited productivity. The solubility of 15-methyl-6-dEB in water was determined to be 0.25-0.40 g/L, although higher titers are possible in fermentation medium. The incorporation of the hydrophobic resin XAD-16HP resulted in both the in situ adsorption of the product and the slow release of the diketide precursor. The resin-containing fermentation achieved 1.3 g/L 15-methyl-6-dEB, 50% higher than the resin-free process. By combining classical mutagenesis, recombinant DNA techniques, and process development, 15-methyl-6-dEB productivity was increased by over 100% in a scalable fermentation process.