长波紫外线与8-甲氧基补骨脂素复合选育天蓝淡红链霉菌(S.coeruleorubidus) 高产株的研究

首次报道长波紫外线(near ultraviole)与8-甲氧基补骨脂素(8-methoxypsoralen)复合选育天蓝淡红链霉菌(S. coeruleorubidus)的研究结果,曾获柔红霉素较出发株稳增6.7%的高产株。     

利用云南红豆杉悬浮细胞生产紫杉醇和紫杉烷类化合物

云南红豆杉(Taxus yunnanensis Cheng et L. K. Fu)的一株紫杉醇高产细胞系经过8年多的继代培养,仍保持较稳定的紫杉烷类化合物的生物合成能力.从此株紫杉醇高产细胞系的悬浮培养物中分离到8个紫杉烷类化合物,经核磁共振光谱和质谱数据分析,它们的化学结构分别是2,5,10-三乙酰氧基-14-丙酰氧基紫杉二烯(1)、 2,5,10-三乙酰氧基-14-(2′-甲基丙酰氧基)紫杉二烯(2)、 2,5,10,14-四乙酰氧基紫杉二烯(3)、 2,5,10-三乙酰氧基-14-(2′-甲基-3′-羟基丁酰氧基)紫杉二烯及其差向异构体(4和5)、巴卡亭Ⅳ(6)、巴卡亭Ⅲ (7)和紫杉醇(8).化合物3、 5-7为首次从云南红豆杉细胞培养物中分离到.定性分析表明,云南红豆杉细胞悬浮培养液中的化学成分与培养细胞中的相似.另外,此株紫杉醇高产细胞系的紫杉醇含量可高达0.3%,可用来进行大规模培养.     

8-甲氧基补骨脂素及其与紫外线复合选育天兰淡红链霉菌的研究

本文比较8-甲氧基补骨脂素(8-Mop)及其与紫外线复合处理柔红霉素产生菌——天兰淡红链霉菌(S．coeruleorubidus)的选育效果,分别获得柔红霉素产率较出发菌株均提高5％以上的高产株,该菌株已在国内应用。     

利用云南红豆杉悬浮细胞生产紫杉醇和紫杉烷类化合物(英文)

云南红豆杉 (TaxusyunnanensisChengetL .K .Fu)的一株紫杉醇高产细胞系经过 8年多的继代培养 ,仍保持较稳定的紫杉烷类化合物的生物合成能力。从此株紫杉醇高产细胞系的悬浮培养物中分离到 8个紫杉烷类化合物 ,经核磁共振光谱和质谱数据分析 ,它们的化学结构分别是 2 ,5 ,10_三乙酰氧基_14_丙酰氧基紫杉二烯 (1)、2 ,5 ,10_三乙酰氧基_14_(2′_甲基丙酰氧基 )紫杉二烯 (2 )、2 ,5 ,10 ,14_四乙酰氧基紫杉二烯 (3)、2 ,5 ,10_三乙酰氧基_14_(2′_甲基_3′_羟基丁酰氧基 )紫杉二烯及其差向异构体 (4和 5 )、巴卡亭Ⅳ (6 )、巴卡亭Ⅲ (7)和紫杉醇 (8)。化合物 3、5 - 7为首次从云南红豆杉细胞培养物中分离到。定性分析表明 ,云南红豆杉细胞悬浮培养液中的化学成分与培养细胞中的相似。另外 ,此株紫杉醇高产细胞系的紫杉醇含量可高达 0 .3% ,可用来进行大规模培养     

8-Mop选育肉桂地链霉菌(StreptomycesCinnamonensis)的研究

作者首次报导8－甲氧基补骨（8－Mop)单一选育肉桂地链霉菌（Streptomyces cinnamonensis)的研究结果,曾有摇瓶发酵产量增幅达7．5％的稳产高产株获得,高产株已在国内应用。     

阿维链霉菌的诱变育种

以阿维链霉菌(Streptomyces avermitilis)76-12为出发菌株,采用亚硝基胍、吖啶橙、紫外线和氯化锂分别对其孢子和原生质体进行诱变,经抗代谢物理性筛选,获得一系列高产突变株,其中N-1-2高产突变株的发酵单位是出发菌株的2.47倍。实验中同时获得了只产阿维菌素a组分的突变株G-32、Bla组分含量高的Ave8菌株和产蓝绿色孢子的突变株UA-G等。     

提高Lovastatin产生菌生物合成能力的研究

用微波等离子体(N^ 20w，4win)诱变M．ruber，筛选到一株高产菌株，再经过发酵工艺优化，最终Lovastaitin的发酵产率提高21．8％。     

重配技术构建高产H1N1型流感疫苗病毒株

目的以经典重配技术制备高产H1N1流感疫苗病毒株。方法以野生型A1/云南昆明/03/2009(H1N1)作为HA及NA基因的供体株,以WHO疫苗株A/Perth/16/2009(H3N2)作为高产基因供体株,共同感染SPF鸡胚,经抗H3及抗N2血清中和筛选法及终末稀释法筛选高产重配H1N1病毒。结果获得一株重配H1N1流感病毒株,病毒血凝滴度为1∶4 096,病毒滴度为7.8 lg EID50/mL,显示为鸡胚高产病毒株;血凝抑制结果为1∶1 024,单向免疫扩散试验结果为阳性,证明抗原性与野生株一致;基因测序结果表明重配株的HA及NA基因序列与野生株序列一致。结论构建了高产重配H1H1流感疫苗病毒株,并应用经典重配技术建立了制备高产流感疫苗病毒株的技术平台。     

8-Mop选育天兰淡红链霉菌(S. Coeruleorubidus)高产株

本文首次报导天兰淡红链霉菌（S.coeruleorubidus)经8－甲氧基补骨脂素选育后,获得一株摇瓶产量稳增7．47％的高产株,已在国内应用。     

捷安肽素高产突变株96孔板筛选方法的建立

建立了一种快速灵敏地筛选出捷安肽素(JAA)高产突变株的方法。主要利用96多孔板固体培养菌体,用50%乙醇为最佳浸提液浸提4h,滤纸片法检测JAA生物活性的点样量为100μL,筛选出5株高产突变株,经摇瓶复筛选出2株高产突变株,经一剂量法检测,其抑菌圈直径较出发菌株提高了14%左右。     

Heterogeneity of ribosomal proteins among Streptomyces species and its application to identification

The ribosomal proteins from 11 Streptomyces strains representing various numerical taxonomic clusters were compared by two-dimensional PAGE. The protein patterns were specific for each species and were unaffected by acridine dye treatment, suggesting genetic stability of ribosomal proteins. An attempt was made to identify one strain of Streptomyces by both traditional taxonomic methods and analysis of the ribosomal protein patterns. Both methods identified the strain as Streptomyces lavendulae, and protein pattern analysis also showed that Streptomyces avidinii was closely related to this species. The practical application of ribosomal protein patterns in Streptomyces taxonomy was therefore demonstrated.     

Polyacrylamide gel electrophoresis analysis of ribosomal protein: a new approach for actinomycete taxonomy.

The ribosomal (r)-proteins from eleven Streptomyces strains representing various numerical taxonomic clusters were compared by two-dimensional polyacrylamide-gel electrophoresis (2D-PAGE). The protein patterns were specific for each species. An attempt was made to identify one strain of Streptomyces by both traditional taxonomic methods and 2D-PAGE analysis of the r-protein patterns. Both methods identified the strain as Streptomyces lavendulae, and protein pattern analysis also showed that S. griseolavendus was a variant of S. lavendulae. Actinomycete r-protein AT-L30 exhibited electrophoretic mobility that is specific for each genus. On the basis of this observation, we analyzed AT-L30 r-proteins from numerous strains of species belonging to the genera Actinomadura, Microtetraspora, Streptosporangium, Nocardia, Rhodococcus and mycolate-less wall chemotype-IV actinomycetes. The results strongly supported the conclusions of previous work and thus proved the efficacy of r-protein analysis as a novel approach for taxonomy of actinomycetes.     

Molecular analysis of beta-lactamases from four species of Streptomyces: comparison of amino acid sequences with those of other beta-lactamases

Genes encoding extracellular beta-lactamases of Streptomyces badius, Streptomyces cacaoi, Streptomyces fradiae and Streptomyces lavendulae were cloned and mapped in Streptomyces lividans. DNA sequence analysis of the beta-lactamase genes revealed a high overall G + C content, ranging from 71 to 75 mol%, with a G + C content of 95 mol% at the third position of the codons for all four genes. The primary structure of the beta-lactamases including their signal peptides was deduced. The four beta-lactamases exhibited homology to each other and to class A beta-lactamases from other bacterial genera. We suggest that Streptomyces beta-lactamases are representatives of a superfamily of genes, from which class A beta-lactamases of Gram-negative bacteria may have evolved.     

Actinomycetes producing trypsin inhibitors

The ability to produce inhibitors of trypsin-like proteases was tested in 300 cultures of actinomycetes freshly isolated from different soils of the USSR. A high antitrypsin activity was found in seven cultures which had not been known before as those producing trypsin inhibitors: Streptomyces sporoclivatus 28 (1), S. lavendulae 29 (4), S. diastatochromogenes 20 (4), S. violascens 52 (8), S. bikiniensis 17 (5), S. filamentosus 32 (11), and Streptoverticillium cinnamoneum 86 (8). The morphological and cultural characteristics of the strains were studied as well as their production of trypsin inhibitors.     

Protein tyrosine phosphorylation in streptomycetes

Using phosphotyrosine-specific antibodies, we demonstrate that in several Streptomyces spp. a variety of proteins are phosphorylated on tyrosine residues. Tyrosine phosphorylation was found in a number of Streptomyces species including Streptomyces lividans, Streptomyces hygroscopicus and Streptomyces lavendulae. Each species exhibited a unique pattern of protein tyrosine phosphorylation. Moreover, the patterns of tyrosine phosphorylation varied during the growth phase and were also influenced by culture conditions. We suggest that metabolic shifts during the complex growth cycle of these filamentous bacteria, and possibly secondary metabolic pathways, may be controlled by the action of protein tyrosine kinases and phosphatases, as has been demonstrated in signal transduction pathways in eukaryotic organisms.     

淡紫灰链霉菌gCLA4坏死诱导蛋白基因的克隆表达及功能

【背景】淡紫灰链霉菌(Streptomyceslavendulae)gCLA4是从黄瓜中分离到的一株放线菌,研究表明该菌对多种植物病原菌均有很好的拮抗作用,具有潜在的生防价值。【目的】深入研究Streptomyces lavendulae gCLA4中坏死诱导蛋白(necrosis-inducing protein) 4955的功能,明确其提高植物抗性的作用机制。【方法】对坏死诱导蛋白4955基因进行克隆,于Escherichia coli BL21(DE3)中表达,并以烟草为材料检测该蛋白的活性和稳定性;使用Protparam、PredictProtein、NCBI CDD、SWISS-MODEL分析蛋白的基本性质和三维结构;检测该蛋白对烟草防御反应相关酶活(CAT、SOD、POD、PAL)和防御相关基因(NPR1、PR1-b、PAL、LOX、PR1-a)表达量的影响。【结果】蛋白4955的耐受温度达40°C,耐受pH 6.0-10.0。该蛋白分子量为24 491.12 Da,由225个氨基酸组成,其等电点为5.96,经氨基酸序列比对含保守NPP1结构域。蛋白4955处理烟草2 d时,烟草CAT、SOD、PAL酶活增加,POD酶活无显著变化。该蛋白处理烟草第1、3、5天时,基因PR1-b、LOX表达量提高;在第4天时,基因PAL的表达量提高。【结论】Streptomyces lavendulae gCLA4中的坏死诱导蛋白4955确实能诱导烟草中的植物防御反应。     

Novel family of cholesterol esterases produced by actinomycetes bacteria

Although cholesterol esterase (CHE; EC 3.1.1.13) is widespread in nature, CHEs from Streptomyces lavendulae and Streptomyces sp. X9 are the only known CHEs produced by actinomycetes. We purified CHEs from S. avermitilis JCM5070, and S. griseus IFO13350 and identified four new CHEs from actinomycetes. The enzymic properties of the CHEs from Streptomyces sp. X9, S. avermitilis, and S. griseus including substrate specificity, sensitivity to inhibitors and optimal conditions for catalysis were similar. We identified genes for the CHEs from Streptomyces sp. X9 and S. avermitilis and the encoded predicted sequences comprised 217 and 214 amino acid residues, respectively, with 64% similarity. The CHEs from Streptomyces sp. X9 and S. avermitilis were also 54 and 57% similar, respectively, to S. lavendulae CHE, indicating that these CHEs are orthologs. Phylogenetic analysis showed that they are distantly related to the conventional lipase/esterase type CHEs from mammals, yeasts and other bacteria. The actinomycetes CHEs did not have the Gly-Xaa-Ser-Xaa-Gly sequence that is conserved in the lipase/esterase family. A database search showed that orthologs of this type of CHE were restricted to actinomycetes. These findings imply that the actinomycetes CHEs constitute a novel family of cholesterol esterases.     

Activation and stabilization of penicillin V acylase from streptomyces lavendulae in the presence of glycerol and glycols

Penicillin V acylase (EC 3.5.1.11) from Streptomyces lavendulae showed both enhanced activity and stability in mixed water/glycerol and water/glycols solvents. The catalytic activity was increased up to a critical concentration of these cosolvents, but further addition of the latter led to a gradual protein deactivation. The highest stabilizing effect was achieved in the presence of glycerol. Thermal stability was increased proportionally to the concentration of glycerol and glycols in the reaction mixture only if the amount added is below the threshold concentration. Reaction conditions that allow simultaneously enhanced activity and stability in the hydrolysis of penicillin V catalyzed by penicillin V acylase from S. lavendulae could be established.     

Characterization of the sat 4 gene encoding a streptothricin acetyltransferase in Campylobacter coli BE/G4

Abstract The sat 4 streptothricin resistance gene from Campylobacter coli BE/G4 was cloned into pUC18, and its nucleotide sequence was determined. Streptothricin acetyltransferase activity was detected in Escherichia coli cells containing recombinant plasmid pAT132 which carries the sat4 gene as an insert. The deduced amino acid sequence displayed 21–27% amino acid identity with streptothricin acetyltransferases from E. coli and streptothricin producers Streptomyces lavendulae and Streptomyces noursei . The sat 4 gene was detected by hybridization in clinical and environmental isolates of Campylobacter spp.     

Self-protection mechanism in D-cycloserine-producing Streptomyces lavendulae. Gene cloning, characterization, and kinetics of its alanine racemase and D-alanyl-D-alanine ligase, which are target enzymes of D-cycloserine

An antibiotic, D-cycloserine (DCS), inhibits the catalytic activities of alanine racemase (ALR) and d-alanyl-d-alanine ligase (DDL), which are necessary for the biosynthesis of the bacterial cell wall. In this study, we cloned both genes encoding ALR and DDL, designated alrS and ddlS, respectively, from DCS-producing Streptomyces lavendulae ATCC25233. Each gene product was purified to homogeneity and characterized. Escherichia coli, transformed with a pET vector carrying alrS or ddlS, displays higher resistance to DCS than the same host carrying the E. coli ALR- or DDL-encoded gene inserted into the pET vector. Although the S. lavendulae DDL was competitively inhibited by DCS, the K(i) value (920 microM) was obviously higher (40 approximately 100-fold) than those for E. coli DdlA (9 microM) or DdlB (27 microM). The high K(i) value of the S. lavendulae DDL suggests that the enzyme may be a self-resistance determinant in the DCS-producing microorganism. Kinetic studies for the S. lavendulae ALR suggest that the time-dependent inactivation rate of the enzyme by DCS is absolutely slower than that of the E. coli ALR. We conclude that ALR from DCS-producing S. lavendulae is also one of the self-resistance determinants.