Isolation,enzyme-bound structure and antibacterial activity of platencin A1 from Streptomyces platensis

Natural products continue to serve as one of the best sources for discovery of antibacterial agents as exemplified by the recent discoveries of platensimycin and platencin. Chemical modifications as well as discovery of congeners are the main sources for gaining knowledge of structure–activity relationship of natural products. Screening for congeners in the extracts of the fermentation broths of Streptomyces platensis led to the isolation of platencin A₁, a hydroxy congener of platencin. The hydroxylation of the tricyclic enone moiety negatively affected the antibacterial activity and appears to be consistent with the hydrophobic binding pocket of the FabF. Isolation, structure, enzyme-bound structure and activity of platencin A₁ and two other congeners have been described.     

Microbial inhibitors of phospholipase C

Inhibitors of the Clostridium perfringens phospholipase C were prepared from the filtrates of the culture liquids of Streptomyces saracetidus and Streptomyces species using soluble and cross-linked polyelectrolytes. The technological scheme of isolation involves ultrafiltration. The inhibitors produced by the two strains had different chemical nature. The preparation obtained from Str. saraceticus was proved to be a complex of inhibitors that were separated by gel-chromatography into a major polypeptide with a molecular weight of 5500-6500 and a low-molecular weight glycopeptide. The inhibitor obtained from Str. species was found to be a high-molecular weight protein.     

棒状链霉菌lat基因的中断对棒酸产量的影响

从棒状链霉菌中克隆1.8kb的lat基因片段,构建了基因置换质粒pXAL1和pXAL2。运用接合转移方法把中断载体导入棒状链霉菌中进行lat的中断,得到1株接合转移子AmrThios,命名为XAL863。通过Southern杂交分析及赖氨酸转氨酶活性测定,证明此菌株的lat基因被中断。通过发酵培养,HPLC方法检测棒酸含量,发现棒酸产量明显提高,约为原产量的1.8倍。     

Interactions of Streptomyces serine-protease inhibitors with Streptomyces griseus metalloendopeptidase II.

Streptomyces griseus metalloendopeptidase II (SGMPII) was shown to form tight complexes with several Streptomyces protein inhibitors which had been believed to be specific to serine proteases, such as Streptomyces subtilisin inhibitor (SSI), plasminostreptin (PS), and alkaline protease inhibitor-2c' (API-2c'), as well as with Streptomyces metalloprotease inhibitor (SMPI). The dissociation constants of complexes between SGMPII and these inhibitors were successfully determined by using a novel fluorogenic bimane-peptide substrate. The values ranged from nM to pM. The results of studies by gel chromatographic and enzymatic analyses indicated that SGMPII is liberated from the complex with SSI by the addition of subtilisin BPN'. SGMPII and subtilisin BPN' proved, therefore, to interact with SSI in a competitive manner, despite the difference in the chemical nature of their active sites.     

不吸水链霉菌梧州新亚种限制-修饰系统初探

采用菌丝体原位包埋方法和高压脉冲电泳技术从不吸水链霉菌梧州新亚种(Streptomyces ahygroscopicus wuzhouensis neosubsp. 11371)中分离得到两条质粒DNA带.通过双向电泳证明,2个质粒均为线性分子,按照分子量大小依次命名为pSAL1、pSAL2.并对不吸水链霉菌梧州新亚种的限制-修饰系统进行初步探讨:将来自变铅青链霉菌TK54的高拷贝质粒pIJ702转化不吸水链霉菌梧州新亚种原生质体,未能得到转化子,改用pIJ702转化不吸水链霉菌梧州新亚种U-3原生质体,得到了转化子.     

Effect of starter unit availability on the spectrum of manumycin-type metabolites produced by Streptomyces nodosus ssp. asukaensis

Aims: Production of minor asukamycin congeners and its new derivatives by combination of targeted genetic manipulations with specific precursor feeding in the producer of asukamycin, Streptomyces nodosus ssp. asukaensis. Methods and Results: Structural variations of manumycins lie only in the diverse initiation of the ‘upper’ polyketide chain. Inactivation of the gene involved in the biosynthesis of cyclohexanecarboxylic acid (CHC) turned off the production of asukamycin in the mutant strain and allowed an increased production of other manumycins with the branched end of the upper chain. The ratio of produced metabolites was further affected by specific precursor feeding. Precursor‐directed biosynthesis of a new asukamycin analogue (asukamycin I, 28%) with linear initiation of the upper chain was achieved by feeding norleucine to the mutant strain. Another asukamycin analogue with the unbranched upper chain (asukamycin H, 14%) was formed by the CHC‐deficient strain expressing a heterologous gene putatively involved in the formation of the n‐butyryl‐CoA starter unit of manumycin A. Conclusions: Combination of the described techniques proved to be an efficient tool for the biosynthesis of minor or novel manumycins. Significance and Impact of the Study: Production of two novel asukamycin derivatives, asukamycins H and I, was achieved. Variations appeared in the upper polyketide chain, the major determinant of enzyme‐inhibitory features of manumycins, affecting their cancerostatic or anti‐inflammatory features.     

Biochemical and Genetic Insights into Asukamycin Biosynthesis

Asukamycin, a member of the manumycin family metabolites, is an antimicrobial and potential antitumor agent isolated from Streptomyces nodosus subsp. asukaensis. The entire asukamycin biosynthetic gene cluster was cloned, assembled, and expressed heterologously in Streptomyces lividans. Bioinformatic analysis and mutagenesis studies elucidated the biosynthetic pathway at the genetic and biochemical level. Four gene sets, asuA–D, govern the formation and assembly of the asukamycin building blocks: a 3-amino-4-hydroxybenzoic acid core component, a cyclohexane ring, two triene polyketide chains, and a 2-amino-3-hydroxycyclopent-2-enone moiety to form the intermediate protoasukamycin. AsuE1 and AsuE2 catalyze the conversion of protoasukamycin to 4-hydroxyprotoasukamycin, which is epoxidized at C5–C6 by AsuE3 to the final product, asukamycin. Branched acyl CoA starter units, derived from Val, Leu, and Ile, can be incorporated by the actions of the polyketide synthase III (KSIII) AsuC3/C4 as well as the cellular fatty acid synthase FabH to produce the asukamycin congeners A2–A7. In addition, the type II thioesterase AsuC15 limits the cellular level of ω-cyclohexyl fatty acids and likely maintains homeostasis of the cellular membrane.     

Ultrastructural studies of sporulation in Streptomyces.

This is the first study of sporogenesis in Streptomyces carried out on a relatively high number of species (seven) which allows us, using also previously published results, to establish a general picture of this process. In the sporogenesis of Streptomyces two basic stages can be considered: the sporulation septum synthesis and the arthrospore maturation. Our ultrastructural study of the sporulation septum formation suggests the existance within this genus of three basic types. Type I is distinguished because the septum is formed from the beginning by two separate cross walls. Within this type we include Streptomyces erythraeus, Streptomyces albus, and Streptomyces aureofaciens and also include Streptomyces venezuelae, Streptomyces griseus, and Streptomyces osteogriseus. Type II is distinguished because there is a deposit of material previous to the synthesis of the double annulus which completes the septum. This type can be divided into two subtypes. In the first the deposits are wedge-shaped and the double annulus is clearly visible, and to this group belong Streptomyces flaveolus, Streptomyces ambofaciens, and Streptomyces coelicolor. In the second the deposits, which have a different shape and are very well developed, constitute almost entirely the sporulation septum in which the double annulus is barely visible; Streptomyces antibioticus and also Streptomyces viridochromogenes belong to this group. Type III, represented by Streptomyces cinnamonensis, is distinguished because the septum is formed by a single cross wall.     

Studies on an Enzyme Capable of Splitting β-d-1,6-Glucosidic Linkage

Examination was made on the morphological and cultural characteristics of the lutease-producing Streptomyces strain No. OP-4-5 isolated from a dust. The strain was identified as Streptomyces griseus. In addition, it was proved that 2 strains of Streptomyces griseus produce lutease in a test for lutease production in Streptomyces species. Streptomyces parvus and Streptomyces niveoruber also produce the same enzyme. However, production of the lutease by these 4 strains was less than that of produced by Streptomyces griseus strain No. OP-4-5 which was isolated by the authors.     

Production of transglutaminase by Streptomyces isolates in solid-state fermentation

Aims:  To screen Streptomyces isolates for transglutaminase (TGase) production in solid-state fermentation (SSF) on various substrates.
Methods and Results:  Streptomyces mobaraensis NRRL B-3729, Streptomyces paucisporogenes ATCC 12596 and Streptomyces platensis NRRL 2364 strains were screened for extracellular TGase production in SSF on different substrates. High-protein-content beans, peas and lentils proved to be the best substrates. Good TGase production was obtained on liver kidney beans and green mung beans in a 4- to 6-day SSF. Temperature optima of the enzymes varied between 45 to 50°C. Molecular weight determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS PAGE) indicated similar size (∼37 kDa) for all three enzymes. TGase was the dominating protein band on SDS PAGE for two Streptomyces strains in SSF extracts. Other enzymes were present in smaller quantities.
Conclusions:  Streptomyces mobaraensis NRRL B-3729, S. paucisporogenes ATCC 12596 and S. platensis NRRL 2364 strains were successfully propagated under SSF conditions on crushed/milled liver kidney bean and green mung bean to obtain good level of TGase.
Significance and Impact of the Study:  Owing to much reduced production cost and direct applicability, SSF TGase without downstream processing (cheap in situ enzyme, crude enzyme) may be an excellent candidate for some nonfood applications.     

Streptomyces sp. Z-11-6--a new producer of extracellular L-glutamate oxidase

Glutamate oxidase activity was studied in 1254 Streptomyces strains isolated from the zonal soils of various regions of Russia and other countries. Seven strains proved to be producers of extracellular L-glutamate oxidase. The most active producer strain was identified, and the conditions of enzyme biosynthesis were optimized. A multistep-mutagenesis and selection procedure allowed a genetically stable strain, Streptomyces sp. Z-11-6, to be obtained, whose glutamate oxidase activity was 40 times higher than that of the original natural isolate.     

同源重组菌株M53的构建及其作为链霉菌通用克隆受体的可行性

为了发展优良的链霉菌宿主系统 ,以带有硫链丝菌素抗性的同源重组葡萄糖异构酶 (GI)缺陷型菌株M10 33XW78,M10 33XW194为出发菌株 ,利用摇瓶、影印和负筛的方法 ,获得一株既无GI活性又对硫链丝菌素敏感的回复菌株 ,命名为淀粉酶链霉菌M5 3.通过染色体PCR检测、酶切图谱鉴定、序列分析等方法 ,确认M5 3含有和M10 33一致的 1 2kb葡萄糖异构酶基因 ,但在结构基因345～ 10 95bp片段内有 17个碱基发生突变 .这说明在染色体内自发同源重组过程中 ,有低频率的突变位点引入 .酶活力测定和SDS PAGE分析表明 ,该突变的GI基因不表达 4 2 5KD葡萄糖异构酶 ,这为M5 3菌株发展成为优良的链霉菌宿主提供了足够的表达空间 .一系列的转化实验也证明了M5 3菌株很可能是一种新型链霉菌克隆受体     

抗生素AGPM发酵过程中蛋白质表达差异的研究

由土壤中筛选到一能产生新型抗肿瘤抗生素AGPM的藤黄灰链霉菌株,应用蛋白质双向电泳方法,比较了藤黄灰链霉菌在发酵24h与72h蛋白质表达的差异,发现在发酵72h有17个蛋白质差异点出现,此外还发现一些蛋白质的含量明显高于24h的同种蛋白含量,这表明这些蛋白可能与抗生素AGPM的合成以及和菌体的生长等有关。     

海洋链霉菌GB-2发酵产物的抗细菌活性及性质研究

从连云港海域潮间带采集的样品中筛选得到一株产高活性抗细菌物质的链霉菌GB-2。该菌的发酵产物对蜡样芽孢杆菌AS1.1846、金黄色葡萄球菌ATCC25923及6株耐药性金黄色葡萄球菌等11株革兰氏阳性菌,大肠杆菌AS1.487、荧光假单胞菌AS1.1802等4株革兰氏阴性菌有显著拮抗作用。纸层析对抗细菌物质分析结果表明,菌株GB-2所产抗细菌物质是中性的水溶性物质,其产生与海水的存在有显著相关性。发酵液稳定性研究表明,该物质在121℃,pH1和pH12条件下抑菌活性均不变;紫外线照射也不影响其抑细菌活性。显示菌株GB-2产物在生防、食品及医药方面潜在的应用价值。     

Four new doramectin congeners with acaricidal and insecticidal activity from Streptomyces avermitilis NEAU1069

Four new doramectin congeners, 1-4, were isolated from Streptomyces avermitilis NEAU1069. The structures of 1-4 were elucidated on the basis of spectroscopic analysis, including 1D- and 2D-NMR as well as HR-ESI-MS, ESI-MS, UV, and IR, and comparison with literature data. All compounds exhibited noticeable acaricidal and insecticidal activities. Especially compound 2 was found to be the most potent pesticide of the compounds evaluated with the IC(50) values of 10.2, 65.1 and 124.4 μg/ml against adult two-spotted spider mites (Tetranychus urticae Koch), two-spotted spider mite eggs, and Mythimna separata, respectively, which are comparable to those of commercial pesticide milbemycin A(3)/A(4) as positive reference.     

Quantitative determination of echinomycin by disc agar diffusion assay

Echinomycin is a peptide antibiotic of the quinoxaline group produced by Streptomyces echinatus. In propitious circumstances it can be determined by ultraviolet spectrophotometry, but for work with analogues and congeners a more specific assay is required. A disc agar diffusion technique has been developed and optimised for this purpose. It yields log dose-response curves which are linear over at least a 20-fold range of antibiotic concentration, and the variation of sensitivity with inoculum size, preincubation time, and temperature of incubation has been investigated. Other quinoxaline antibiotics can be assayed by the same technique; they yield quite different log dose-response curves indicative of lower potency associated with triostins as compared to quinomycins. The application of the technique to follow echinomycin production by S. echinatus A8331 in culture in maltose minimal medium is described.     

Enhancement of Lignin Degradation in Streptomyces spp. by Protoplast Fusion

Protoplast fusion was investigated as a technique for genetically manipulating two lignin-degrading Streptomyces strains, Streptomyces viridosporus T7A and Streptomyces setonii 75Vi2. Four of 19 recombinants tested showed enhanced production of acid-precipitable polymeric lignin (APPL), producing 155 to 264% more APPL from corn stover lignocellulose than was produced by the wild-type S. viridosporus T7A. APPLs are lignin degradation intermediates known to be potentially valuable chemical products produced by bioconversion of lignin with Streptomyces spp. The prospects of utilizing protoplast fusion to construct APPL-overproducing Streptomyces strains was considered especially promising.     

<Emphasis Type="Italic">Streptomyces roseoalbus</Emphasis> sp. nov., an actinomycete isolated from soil in Yunnan,China

A strain YIM 33098T (= CCTCC AA001027T = DSM 41831T) was isolated from a forest soil sample collected from Nanning in Guangxi Province, China, in the course of screening for producers of new drug lead compounds. This strain was identified by using a polyphasic approach. The results showed that it should be assigned to the genus Streptomyces. An almost complete 16S rRNA gene sequence of the strain was determined and compared with those of representative Streptomyces species. Strain YIM 33098T was clustered in the same subclade with Streptomyces tendae ATCC19812T and Streptomyces eurythermus ATCC14975T. Similarities of strain YIM 33098T with the two strains were 97.35% and 97.42%, respectively. Based on the phenotypic and genotypic evidence, it is therefore proposed that strain YIM 33098T should be classified in the genus Streptomyces as a new species under the name of Streptomyces nanningensis sp. nov.     

链霉菌Streptomyces sp.KIB-H1424中的一个新的烷基间苯二酚类化合物

运用色谱学方法对一株来自云南省玉溪市元江县的土壤链霉菌Streptomyces sp.KIB-H1424的次级代谢产物进行分离纯化,得到6个单体化合物。运用NMR、MS及与文献数据对比等手段,确定其为一系列的烷基间苯二酚类似物,其中包括1个新的烷基间苯二酚类化合物Adiposatatin E(1)以及5个已知的烷基间苯二酚类似物Adipostatin A(2)、Adiposatin B(3)、Adipostatin C(4)、Adipostatin D(5)和5-Heptadecyl-1,3-benzenediol(6)。运用滤纸片法测定6个化合物对几种病原细菌和真菌的抑菌活性,发现化合物1~6不具有显著的抑菌活性。