南昌霉素高产菌株的链霉素抗性基因突变诱变筛选研究

通过对链霉素对南昌霉素（Nanchangmycin)产生菌NS－41－80菌株孢子的致死浓度测定基础上,采用诱变剂甲基磺酸乙酯（EMS）的不同诱发剂量对菌株孢子进行诱变处理,诱变处理的孢子涂布在含链霉素（10ug/mL)致死浓度的高氏平板上,获得了大量的链霉素抗性基因（str)突变株。然后从3,000株链霉素抗性基因（str)突变株中通过初筛获得比诱变出发菌株产素能力提高20％以上的菌株202株,再进一步通过摇瓶复筛,获得比出发菌株产素能力分别提高100％,200％,300％高产菌株为48株,7株和1株,分别为复筛菌和初筛菌株的23．76％和1．60％,3．46％和0．23％,0．5％和0．03％,将产素能力提高240％以上5个菌株连同出发菌株连续3批次进行摇瓶发酵结果,5个突变株的产素能力均比出发菌株的产素能力提高57％－96．4％,其中突变株80－5．3－165菌株摇瓶发酵单位达6,000ug/mL以上,3批次摇瓶平均发酵单位达5,855ug/mL,建立了南昌霉素高产菌株的链霉素抗性基因突变诱变快速高效的筛选方法。     

酿酒酵母和产朊假丝酵母原生质体的形成、再生及属间融合

使用由亚硝基胍诱变所得到的营养缺陷型作为单倍体融合亲株的核基因标记,同时也采用线粒体球红霉素抗性突变株的小菌落形式作为融合亲株的线粒体基因标记。酿酒酵母(Saccharomyces cerevisiae)和产朊假丝酵母(Candida utilis)两亲株原生质体的制备是用对数生长早期的细胞在蜗牛酶和0.7M KCl及β-巯基乙醇或二巯基苏糖醇的作用下完成的。二者的原生质体的形成率在30—60分钟内达到90—99％。原生质体再生率,酿酒酵母最高为29—35％,产朊假丝酵母为7.5％。两亲株的原生质体在35％PEG(M.W.6,000),10mM CaCl_2条件下被诱导融合。在基础培养基上,长出以营养互补为标记的融合菌株。融合频率为10~(-5)—10~(-6)。试验表明,这些融合菌株具有杂种的性质。其中一株杂合子在同化D-木糖、纤维二糖等的能力上比亲株明显增强。     

微波诱变结合抗性突变筛选放线菌素D高产菌株

采用微波结合链霉素抗性筛选法选育放线菌素D的高产菌株。通过考察链霉素对Streptomyces rubiginosohelvolus FIM-N31菌株孢子生长情况的影响确定链霉素致死浓度,出发菌株FIM-N31的孢子经微波辐照处理后,涂布在含链霉素致死浓度（50 μg/mL）的培养基平板上培养,获得了大量的链霉素抗性基因突变株。摇瓶发酵筛选突变株,结果获得一株遗传性状稳定的放线菌素D高产菌Str186,其产放线菌素D的能力比出发菌株提高了8倍以上。     

酿酒酵母缺陷型原生质体的形成再生及融合条件的探讨

营养缺陷型作为酿酒酵母(Saccharomyces Cerevisiae)单倍体融合亲株的遗传标记。采用营养缺陷型LY—2和LY—4菌侏的对数生长前期的细胞,在0.5％的蜗牛酶作用时间40分钟,0.7MKCl高渗稳定剂的条件下,制备原生质体。两亲株的原生质体形成率分别为98.2％和91.0％。原生质体再生率为6.12％和2.13％。两亲株的原生质体在35％聚乙二醇(PEG, M.W.6000),50mMCaCl_2下诱导融合15分钟,在基本培养基上长出营养互补的融合菌株,融合频率为4.14x10_(-4)。本文就两亲株的原生质体形成和再生条件进行了初步的探讨,并进行了原生质体融合的尝试,     

酵母融合菌株的构建及其特性研究

以酿酒酵母和糖化酵母为亲本,通过正交实验优化了亲本原生质体的制备,再生以及融合的最佳条件,由此获得了既具有糖化能力又可产高浓度酒精的稳定的酵母融合菌株。融合株的体积较亲株大,ＤＮＡ含量与两亲株ＤＮＡ含量之和相当,淀粉水解能力,葡萄糖发酵效率,生长速率及产酒精能力等均优于亲株     

用原生质体融合技术选育2—酮基—L—古龙酸高产菌株的研究

对革兰氏阳性的地衣芽孢杆菌（Ｂａｃｉｌｌｕｓｌｉｃｈｅｎｉｆｏｒｍｉｓ）Ｈ１９和革兰氏阴性的２－酮基－Ｌ－古龙酸产生菌Ｓ１９的原生质体的制备条件进行了研究,并采用聚乙二醇作诱导剂进行了两菌株的原生质体融合,用链霉素作为抗性标记对融合子进行了选择。从１７株产生２－酮基－Ｌ－古龙酸的融合子中选出了一株连续传代八次产酸高且产量稳定的融合子１５号。融合子１５号具有两个亲本菌株所具有的一些特性。     

荧光标记金针菇原生质体融合

以金针菇（Flmmulina velutipes）为材料,经异硫氨酸荧光素（FIC）标记的金针菇单核W19菌株的原生质体与未经标记的单核Y7菌株的原生质体在聚乙二醇（PEG）的诱导下进行融合,利用显微操纵仪直接挑取一个带有荧光而另一个不具荧光的原生质体对,培养后,根据融合形成的汉核菌丝具有“锁状连合”这一形态学特征选择、鉴定融合菌株,酯酶同工酶电泳分析结果表明,融合菌株具双亲互补酶带。经测定,多数融合菌株菌丝生长速度和生物量较亲株高,且出菇正常。     

链霉素生产菌—灰色链霉菌和热灰紫链霉菌的原生质体融合的研究

本文采用原生质体融合技术,把链霉素生产菌——灰色链霉菌No．45 Streptomyces griseus No.45(Lin,Rif‘)同耐高温的不产生抗生素的热灰紫链霉菌T272 Strep-griseus thermogriseoviolaceus T272(Lin^s,Rif^f)进行了原生质体融合。以抗性为选择标记,以PEG6000为助融剂,选出了融合体。制备超薄切片后在电镜下观察了原生质体融合的详细过程。在链霉素生物合成受抑制的高温(37℃)下,测定了融合体的抑菌括性,从形态上与两亲株不同的46株融合体中发现6．3％的融合体既有耐高温的特性也有抑菌的活性。     

产谷氨酸棒杆菌B9和T6—13的原生质体融合

棒杆菌Ｂ９和Ｔ６—１３两菌株经ＵＶ变处理得到Ｂ９—２（ＳｍＲ）和Ｔ６—１３—３（ＲｉｆＲ）两菌株,以此两菌株做为出发菌株。将对数前期的培养细胞经青霉素予处理及酶解制备原生质体,用４０％ＰＥＧ６０００为助融剂,进行原生质体融合。用间接法检出具有Ｓｍ、Ｒｉｆ抗性的融合子。融合频率为６．５５×１０－６－１．６４×１０－５,融合子双抗性稳定,产谷氨酸,经摇瓶实验筛选出一株产谷氨酸明显高于亲本的融合子ｆｕ３６。     

林青链霉菌双亲灭活原生质体融合的研究

分别以紫外线、热灭活性林肯链霉菌947和9502原生质体,然后进行灭活双亲的原生质体融合,从16株融合子筛选到林青霉素高产株。用双亲的互补营养缺陷型对林肯链霉菌原生质体的制备、融合、再生的部分条件进行了研究。发现含0．4％Gly和34％蔗糖的SM培养基最适于实验菌株原生质体的制备、再生。聚乙二醇（PEG）分子量对原生质体融合影响不大,其在P缓冲液中的浓度却很重要。含50％PEG的P缓冲液最有利于原生质体融合。     

Screening, mutagenesis and protoplast fusion of Aspergillus niger for the enhancement of extracellular glucose oxidase production

Various strains of Aspergillus niger were screened for extracellular glucose oxidase (GOD) activity. The most effective producer, strain FS-3 (15.9 U mL^–1), was mutagenized using UV-irradiation or ethyl methane sulfonate. Of the 400 mutants obtained, 32 were found to be resistant to 2-deoxy d-glucose, and 17 of these exhibited higher GOD activities (from 114.5 to 332.1%) than the original FS-3 strain. Following determination of antifungal resistance of the highest producing mutants, four mutants were selected and used in protoplast fusions in three different intraspecific crosses. All fusants showed higher activities (from 285.5 to 394.2%) than the original strain. Moreover, of the 30 fusants isolated, 19 showed higher GOD activity than their corresponding higher-producing parent strain.     

质粒消除对冠菌素产生菌T1828生物学效应的影响

采用十二烷基硫酸钠（SDS）和提高生长温度结合法消除T1828菌株质粒后,筛选到无质粒突变株ZWL-15。以原始亲株T1828和无质粒突变株ZWL-15为出发菌株,考察了两者部分生物学特性。结果表明,菌株ZWL．15生长速率快于T1828,进入对数期和稳定期的时间分别提前1h和4h。在菌株ZWL-15发酵过程中添加0．01％的SDS有利于冠菌素的合成,最适发酵温度比T1828升高5℃,达到35℃,发酵周期提前4h,COR相对值是T1828的两倍左右。ZWL-15传代实验表明该突变株很稳定,且对氨苄青霉素敏感,氨苄青霉素基因初步定位于该菌属质粒上。     

在中国卢龙县发现G5型人A组轮状病毒

轮状病毒是引起我国儿童重症腹泻的主要病原。按照WHO轮状病毒监测方案,对2003年间河北省卢龙县开展了以医院和社区为基础的小于5岁儿童轮状病毒腹泻的监测,发现一株新型轮状病毒。该病毒用传统分型引物(G1、G2、G3、G4)扩增不出条带,对其VP7基因全序列测定和分析后确定该毒株为G5型。这是我们在亚洲首次发现的人类G5型轮状病毒。该毒株与猪的G5型C134毒株核苷酸和氨基酸序列同源性分别为88·6%和95·4%,与非洲发现的人的G5型毒株MRC3105核苷酸和氨基酸的同源性为89·9%和94%,与巴西发现的IAL-28毒株核苷酸和氨基酸的同源性为87·2%和93·3%。系统发生树分析表明:卢龙毒株LL36755与其他已经报告的两种猪和一种人类的G5型毒株可能具有相同的起源。这是人轮状病毒G5型首次在亚洲国家发现,而且该毒株可能是由人类轮状病毒与动物轮状病毒毒株自然重组产生。     

Secretory production of recombinant protein by a high cell density culture of a protease negative mutant Escherichia coli strain

Several protease negative mutant strains including HM114, HM126, and HM130 as well as their parent strain KS272 were compared for their growth and secretory production of a model fusion protein, protein A-beta-lactamase. HM114, a strain deficient in two cell envelope proteases, grew slightly faster and produced more fusion protein than the other strains deficient in more proteases. HM114 was grown to a cell dry weight of 47.86 g/L in 29 h using pH-stat, fed-batch cultivation. The beta-lactamase activity was 11.25 x 10(4) U/L, which was 30% higher than that obtained with its parent strain KS272. Up to 96% of protein A-beta-lactamase fusion protein could be recovered by a simple cold osmotic shock method. The specific beta-lactamase activity obtained with HM114 after fractionation was 4.5 times higher than that obtained with KS272.     

Near ultraviolet light inactivation of dihydroxyacid dehydratase in Escherichia coli

The effects of near ultraviolet (NUV) light on a NUV chromophore-containing oxidant-sensitive enzyme, dihydroxyacid dehydratase (DHAD), were measured in seven strains of Escherichia coli. The strains differed in production of the oxidant-defense enzymes, superoxide dismutases (Fe-SOD and Mn-SOD), and catalases HPI and HPII. With the stress of aerobic growth but without NUV exposure, the strains lacking either Fe or Mn SOD or both SODs had 57%, 25%, and 12%, respectively, of the DHAD-specific activity of the parent (K12) strain. Under the same conditions, the catalase strains that were wild type, overproducing, and deficient had comparable DHAD-specific activities. When aerobic cultures were exposed for 30 min to NUV with a fluence of 216 J/m²/s at 310–400 nm, the percentage decreases in DHAD-specific activities were similar (ranging from 75% to 89%) in strains with none, either, or both SODs missing, and in the catalase-overproducing strain. However, the decreases were only 58% and 52% in the strain with catalase missing and in its parent, respectively. The NUV-induced loss of DHAD enzyme activity was not accompanied by any detectable loss of the DHAD protein as measured by polyclonal antibody to DHAD.     

Screening and construction of Saccharomyces cerevisiae strains with improved multi-tolerance and bioethanol fermentation performance

In this study, a systemic analysis was initially performed to investigate the relationship between fermentation-related stress tolerances and ethanol yield. Based on the results obtained, two elite Saccharomyces cerevisiae strains, Z8 and Z15, with variant phenotypes were chosen to construct strains with improved multi-stress tolerance by genome shuffling in combination with optimized initial selection. After three rounds of genome shuffling, a shuffled strain, YZ1, which surpasses its parent strains in osmotic, heat, and acid tolerances, was obtained. Ethanol yields of YZ1 were 3.11%, 10.31%, and 10.55% higher than those of its parent strains under regular, increased heat, and high gravity fermentation conditions, respectively. YZ1 was applied to bioethanol production at an industrial scale. Results demonstrated that the variant phenotypes from available yeast strains could be used as parent stock for yeast breeding and that the genome shuffling approach is sufficiently powerful in combining suitable phenotypes in a single strain.     

Manipulating the pyruvate dehydrogenase bypass of a multi-vitamin auxotrophic yeast Torulopsis glabrata enhanced pyruvate production

AIMS: To investigate the relationship between the activity of pyruvate dehydrogenase (PDH) bypass and the production of pyruvate of a multi-vitamin auxotrophic yeast Torulopsis glabrata. METHODS AND RESULTS: Torulopsis glabrata CCTCC M202019, a multi-vitamin auxotrophic yeast that requires acetate for complete growth on glucose minimum medium, was selected after nitrosoguanidine mutagenesis of the parent strain T. glabrata WSH-IP303 screened in previous study [Li et al. (2001) Appl. Microbiol. Biotechnol. 55, 680-685]. Strain CCTCC M202019 produced 21% higher pyruvate than the parent strain and was genetically stable in flask cultures. The activities of the pyruvate metabolism-related enzymes in parent and mutant strains were measured. Compared with the parent strain, the activity of pyruvate decarboxylase (PDC) of the mutant strain CCTCC M202019 decreased by roughly 40%, while the activity of acetyl-CoA synthetase (ACS) of the mutant increased by 103.5 or 57.4%, respectively, in the presence or absence of acetate. Pyruvate production by the mutant strain CCTCC M202019 reached 68.7 g l(-1) at 62 h (yield on glucose of 0.651 g g(-1)) in a 7-l jar fermentor. CONCLUSIONS: The increased pyruvate yield in T. glabrata CCTCC M202019 was due to a balanced manipulation of the PDH bypass, where the shortage of cytoplasmic acetyl-CoA caused by the decreased activity of PDC was properly compensated by the increased activity of ACS. SIGNIFICANCE AND IMPACT OF THE STUDY: Manipulating the PDH bypass may provide an alternative approach to enhance the production of glycolysis-related metabolites.     

基因组改组技术选育耐酸性琥珀酸放线杆菌

以琥珀酸产生菌Actinobacillus succinogenes CGMCC 1593为出发菌,分别经过紫外线-甲基磺酸乙酯(UV-EMS)和紫外线-硫酸二乙酯(UV-DES)诱变处理,得到7株耐酸性有所提高的突变株.以此作为候选菌库,经3轮原生质体递进融合,筛选获得4株可以在pH 5.6下生长的改组菌株.其中改组菌株F3-21在pH 5.6的完全液体培养基中生长的OD值是原始菌的7倍,在pH 5.2条件下仍能生长;其摇瓶发酵48h琥珀酸产量较原始菌株提高48%.在5L发酵罐中进行分批发酵,当控制pH在较低值(5.6～6.0)时,F3-21厌氧发酵48h积累琥珀酸38.1g/L,较出发菌株提高了45%;当控制pH在6.5～7.0时,F3-21厌氧发酵32h积累琥珀酸40.7g/L.F3-21在5L发酵罐中进行补料分批发酵,厌氧发酵72h,产琥珀酸达67.4g/L.结果说明基因组改组技术能够改进琥珀酸放线菌的耐酸性能及其琥珀酸的产量.     

木霉突变体T1010变异特性分析

木霉突变体T1010与出发菌株实验比较,其生长速度、产孢量、菌丝重量分别提高13．62％、48．52％、29．03％;T1010对培养液中氨态氮的吸收率比出发菌株仍2提高37．09％,T1010对无机磷的吸收率比出发菌株提高15．17％,T1010对K’吸收率比出发菌株高16．49％,在pH值为7．34的培养液中培养3d后,T1010的pH值为5．77,出发菌株的pH值为6．67;突变体T1010对培养液中葡萄糖的吸收能力比出发菌株提高19．64％,突变体T1010利用培养液中的氨基酸量较少,比出发菌株降低18．26％。拮抗作用结果显示,T1010对病菌瓜萎蔫镰刀菌抑制作用、覆盖作用分别比出发菌株提高87．45％、57．38％。