报告基因法比较两种放线菌启动子的活性

摘要：【目的】比较启动子Psf与红霉素抗性基因启动子（PermE*）在链霉菌中的表达强度差异。【方法】本文利用卡那霉素抗性梯度以及邻苯二酚2,3-双加氧酶显色系统,比较了两个启动子的表达差异。【结果】两个启动子在棒状链霉菌(Streptomyces clavuligerus) NRRL3585、天蓝色链霉菌（Streptomyces coelicolor）M145,委内瑞拉链霉菌（Streptomyces venezuelae）ISP5230及变铅青链霉菌（Streptomyces lividans TK     

链霉菌真核型丝氨酸/苏氨酸蛋白激酶的研究进展

简要回顾了丝/苏氨酸蛋白激酶在链霉菌中的发现过程,详细介绍了链霉菌中几个研究较为深入的丝/苏氨酸蛋白激酶的功能,同时对天蓝色链霉菌Streptomyces coelicolor A3(2)和除虫霉菌Streptomyces avermitilis MA-4680中的丝/苏氨酸蛋白激酶的跨膜种类和蛋白结构域特点作了初步的生物信息学分析.     

力达霉素产生菌球孢链霉菌C-1027中atrA同源基因的克隆及分析

目的：在天蓝色链霉菌Streptomyces coelicolor A3(2)中多效性调节因子AtrA（AtrA-c）可通过激活放线紫红素途径特异性的调节因子ActII-ORF4的转录来控制放线紫红素的产生。在灰色链霉菌Streptomyces griseus NBRC13350和阿维链霉菌Streptomyces avermitilis MA-4680中也发现了AtrA-c 编码基因（atrA-c）的同源基因,分别影响链霉素和阿维菌素的生物合成。本文目的在于探索球孢链霉菌C-1027（Streptomyces globisporus C-1027）中是否存在AtrA,克隆球孢链霉菌C-1027中atrA基因并进行生物信息学分析,为进一步确定其对力达霉素产生的调控作用及调控机制奠定基础。[方法] 采用在球孢链霉菌C-1027中异源表达AtrA-c,来确定AtrA-c对力达霉素产量的影响;通过Southern blot 分析来判断在球孢链霉菌C-1027 基因组中是否有atrA-c同源基因;PCR扩增方法获得球孢链霉菌C-1027 atrA基因（atrA-gl）并测序;通过多种生物信息学软件来分析atrA-gl及其与旁侧基因的组织结构、对已发现的AtrA蛋白进行同源性比对及亲缘关系分析。[结果]在球孢链霉菌C-1027中异源表达天蓝色链霉菌AtrA-c蛋白,发现其对力达霉素的产量有影响。以atrA-c为探针,通过Southern blot分析显示球孢链霉菌C-1027基因组中存在atrA-c的同源基因。PCR扩增得到球孢链霉菌C-1027 的atrA基因的全序列以及该基因上下游的旁侧序列（GenBank/EMBL/DDBJ 登录号GU723707）。通过对球孢链霉菌C-1027、天蓝色链霉菌A3(2)、灰色链霉菌NBRC13350以及阿维链霉菌MA-4680 AtrA蛋白序列进行同源性分析发现,四种AtrA蛋白编码氨基酸序列一致性达到65%至 87%,相似性高达70% 至89%。并且,球孢链霉菌C-1027 atrA基因与相邻基因形成的组织结构与天蓝色链霉菌和灰色链霉菌完全一致。根据蛋白质同源性绘制进化树,发现球孢链霉菌AtrA蛋白与灰色链霉菌AtrA蛋白亲缘关系最近。[结论]确定在球孢链霉菌C-1027中存在atrA同源基因并影响力达霉素的产量,克隆了首个力达霉素生物合成基因簇外的调节基因--atrA基因,通过生物信息学分析初步推测了该基因的功能,为进一步研究AtrA-gl对力达霉素途径特异性级联调控网络的调控关系奠定了基础。     

对羟基扁桃酸合酶基因的克隆表达及催化特性

【目的】比较两种不同来源基因重组的对羟基扁桃酸合酶(HmaS),考察其在大肠杆菌中的表达效率。【方法】分别对东方拟无枝酸菌(Amycolatopsis orientalis)和天蓝色链霉菌(Streptomyces coelicolor)来源的hmas进行异源表达,经离子交换层析和凝胶过滤色谱分离纯化获得HmaS,并检测HmaS的酶活和催化特性。【结果】来源于S.coelicolor的HmaSSC2比酶活是来源于A.orientalis的3.6倍;来源于A.orientalis的HmaSAO最适反应温度为28°C,在弱碱性条件下的酶活稳定性较好;来源于S.coelicolor的HmaSSC2最适反应温度为35°C,在28-45°C内保持较高的酶活,具有良好耐热性,在pH 7.0左右酶活最高,更易在偏中性的条件下发挥功能。【结论】HmaSSC2更适用于代谢工程改造大肠杆菌发酵法生产扁桃酸。     

一株Sanguibacter sp.C4产几丁质酶基因的克隆与表达

Chi58是Sanguibacter sp．strain C4产生的一种胞外几丁质酶。通过chiA的特异性PCR引物探测到菌株C4中存在几丁质酶,并将扩增到的几丁质酶基因片段（chiA-F）克隆、测序后,提交GenBank数据库进行同源性搜索。对从GenBank中获得的高同源性序列进行比对,并根据保守区域设计2对PCR引物进行嵌套PCR,扩增出Chi58基因的开放阅读框（ORF）。测序结果表明该酶的ORF由1692个核苷酸组成,编码563个氨基酸,在N端有23个氨基酸的信号肽,其成熟蛋白的分子量应为58．544kDa。对其推导氨基酸的序列分析表明Chi58与沙雷氏菌的几丁质酶（如徂）有高度同源性（88．9％-99．6％）,其结构主要包括信号肽序列、PKD结构域和18家族糖苷水解酶结构域。将该基因克隆到pET32a（＋）载体构建重组质粒pChi58,转入大肠杆菌BL-21（DE3）进行融合表达。经IPTG诱导后,可见分子量约81．1kDa的融合蛋白的表达。     

大肠杆菌-链霉菌穿梭表达载体pMF的构建及其应用

【目的】构建能定点整合到链霉菌(Streptomyces)染色体上的高效表达载体。【方法】以链霉菌自杀型表达载体pLSB2为基础,通过插入链霉菌噬菌体ΦC31整合酶基因int和attP位点(Phage attachment site),构建了能在大肠杆菌和链霉菌之间进行接合转移并定点整合到链霉菌染色体上的表达载体pMF。将pMF转化大肠杆菌ET12567(pUZ8002),并分别接合转移天蓝色链霉菌(Streptomyces coelicolorM145)、变铅青链霉菌(Streptomyces lividansTK24)和红色糖多孢菌(Saccharopolyspora erythraea2338),挑取接合子进行PCR和Southern杂交检测。将来自刺糖多孢菌S08-4的S-腺苷甲硫氨酸合成酶基因(SAM-s)克隆到载体pMF的启动子下游,接合转移到天蓝色链霉菌中。【结果】表明pMF成功整入链霉菌染色体,并且检测到目的蛋白的表达。【结论】构建的pMF载体可作为外源基因定点整合表达的有效工具,为后续的基因功能研究以及链霉菌的遗传改造奠定了基础。     

桑氏链霉菌几丁质酶ChiKJ40基因的克隆表达及其抑菌作用

【背景】目前关于桑氏链霉菌(Streptomyces sampsonii)生防基因的研究不多,仅从其基因组中克隆了2个几丁质酶基因片段,其单个几丁质酶的完整基因序列相关研究未见报道。【目的】克隆S.sampsonii KJ40的几丁质酶基因Chi KJ40并进行原核表达,纯化重组蛋白并研究其抑菌作用。【方法】采用PCR扩增法从S.sampsonii KJ40中克隆几丁质酶基因Chi KJ40,连接到表达载体p ET-32a,导入Escherichia coli BL21(DE3)进行诱导表达。使用His标记蛋白质微量纯化试剂盒对重组几丁质酶进行纯化,Bradford蛋白浓度测定试剂盒测定粗酶液和纯化酶液的浓度,几丁质酶试剂盒测定粗酶液和纯化酶液的几丁质酶活性。观察重组几丁质酶对桉树焦枯病菌(Cylindrocladium scoparium)、栗疫病菌(Cryphonectria parasitica)、链格孢菌(Alternaria alternate)、紫丝核菌(Rhizoctonia violacea)几种致病真菌的抑菌作用。【结果】Chi KJ40基因(登录号为MF434484)在E.coli中经IPTG诱导表达,获得42 k D的重组几丁质酶,不同浓度IPTG在37°C诱导3 h,蛋白产量无明显变化。0.2 mmol/L IPTG 16°C诱导过夜,重组几丁质酶主要以可溶性形式存在于上清,小部分以包涵体存在于沉淀中。粗酶液几丁质酶活性为0.080 U/m L,酶比活力为0.041 U/mg,纯化酶液几丁质酶活性为0.046 U/m L,酶比活力为0.115 U/mg,纯化倍数为2.8,酶活回收率为57.5%。重组几丁质酶处理后,C.scoparium、C.parasitica和A.alternata菌丝细胞出现分节、膨胀,R.violacea菌丝溶解且部分被破坏成碎片。【结论】Chi KJ40基因的研究补充了S.sampsonii的生防背景,为几丁质酶基因找到了新的来源,并为其应用奠定了理论基础。     

两株海洋放线菌的产色素特性

【背景】生物产生的天然色素在工业、农业和纺织工业上有潜在应用价值。【目的】通过对产色素海洋放线菌的分离与筛选,为开发细菌所产生的天然色素在纺织与食品工业中的应用奠定基础。【方法】筛选胞外产可溶性色素的放线菌,并通过16S rRNA基因序列测定和分析构建其系统发育树,并对影响色素产量的因素和色素稳定性进行实验。【结果】获得了一株产蓝色色素的放线菌Q2N-42和一株产黄绿色色素的放线菌X4C-5,16S rRNA基因序列分析表明它们分别与天蓝色链霉菌(Streptomyces coelicolor)或紫红链霉菌(Streptomyces violaceoruber)和普拉滕斯链霉菌(Streptomyces pratensis)的16S rRNA基因序列存在较高的相似性。通过研究不同碳源和氮源对放线菌Q2N-42和X4C-5产色素的影响,发现甘油和硝酸钠能明显地增加天蓝色链霉菌(S. coelicolor) Q2N-42的色素产量,而淀粉和硝酸钠能明显地增加普拉滕斯链霉菌(S. pratensis) X4C-5的色素产量。这两种色素对不同浓度的氧化剂、还原剂、盐和pH都表现出良好的稳定性;色素的红外光谱测定表明,其结构分别包含了?OH、?CH3和C=C基团。通过色素和不同媒染剂对棉线染色,发现硫酸铜是蓝色色素良好的媒染剂,毛线着色较深,洗涤不易脱色;而硫酸亚铁是绿色色素良好的媒染剂,毛线着色较深、有光泽,洗涤不易脱色。【结论】放线菌株Q2N-42和X4C-5所产生的色素为其工业应用提供了潜在的天然色素资源。     

抗生素

<正>产生红霉素的红链霉菌3038菌株分离出质粒pPC7和pPC8已有报道,并给出了限制性内切酶的图谱。该菌株在含有甘氨酸的TSB培养基中,在振荡下于30℃培养18-24小时。在相同的培养基中培养40小时就形成原生质体。按以前的方法进行蓝链霉菌(Streptomyces coelicolor)和红链霉菌属的转化。把原生质体加进含有酵母提取物的R_2培     

利用天蓝色链霉菌转化7-木糖紫杉烷的研究

紫杉醇是目前临床治疗癌症的一线化疗药物,资源紧张,价格昂贵。7-木糖-10-去乙酰基紫杉醇(7-XDT)在红豆杉中含量可达紫杉醇的10倍,脱除木糖基后生成的10-脱乙酰紫杉醇(10-DAT)经乙酰化可生成紫杉醇。通过木聚糖平板对不同菌株进行筛选,从52株供试微生物中,发现27株在木聚糖平板上生长良好。经转化实验筛选,发现一株天蓝色链霉菌(Streptomyces coelicolor YUCM 410115)具有转化7-XDT为10-脱乙酰紫杉醇的能力。菌体细胞经破碎离心后,沉淀及上清液均无转化反应出现,而发酵液的硫酸铵沉淀物则可以转化7-XDT生成10-DAT,表明该菌株能产生一种胞外紫杉醇-7-木糖苷酶,发酵液酶活为6 268U。首次发现天蓝色链霉菌能够产生紫杉醇-7-木糖苷酶,为7-XDT转化生产紫杉醇提供了新的酶源。     

Comparison of enzymatic and antifungal properties between family 18 and 19 chitinases from S. coelicolor A3(2)

Streptomyces coelicolor A3(2) has 13 chitinase genes encoding 11 family 18 and two family 19 chitinases. To compare enzymatic properties of family 19 chitinase and family 18 chitinases produced by the same organism, the four chitinases (Chi18bA, Chi18aC, Chi18aD, and Chi19F), whose genes are expressed at high levels in the presence of chitin, were produced in Escherichia coli and purified. The effect of pH on the hydrolytic activity was very different not only among the four chitinases but also among the substrates. The hydrolytic activity of Chi19F, family 19 chitinase, against soluble substrates was remarkably high as compared with three family 18 chitinases, but was the lowest against crystalline substrates among the four chitinases. On the contrary, Chi18aC, a family 18-subfamily A chitinase, showed highest activity against crystalline substrates. Only Chi19F exhibited significant antifungal activity. Based on these observations, the roles of family 19 chitinases are discussed.     

High-multiplicity of chitinase genes in Streptomyces coelicolor A3(2).

Six different genes for chitinase from ordered cosmids of the chromosome of Streptomyces coelicolor A3(2) were identified by hybridization, using the chitinase genes from other Streptomyces spp. as probes, and cloned. The genes were sequenced and analyzed. The genes, together with an additional chitinase gene obtained from the data bank, can be classified into either family 18 or family 19 of the glycosyl hydrolase classification. The five chitinases that fall into family 18 show diversity in their multiple domain structures as well as in the amino acid sequences of their catalytic domains. The remaining two chitinases are members of family 19 chitinases, since their C-terminus shares more than 70% identity with the catalytic domain of ChiC of Streptomyces griseus, the sole gene for family 19 chitinase so far found in an organism other than higher plants.     

Sequential deletion of all the polyketide synthase and nonribosomal peptide synthetase biosynthetic gene clusters and a 900-kb subtelomeric sequence of the linear chromosome of Streptomyces coelicolor

Streptomyces coelicolor, with its 8 667 507-bp linear chromosome, is the genetically most studied Streptomyces species and is an excellent model for studying antibiotic production and cell differentiation. Here, we report construction of S. coelicolor derivatives containing sequential deletions of all the 10 polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters and a 900-kb subtelomeric sequence (total c. 1.22 Mb, 14% of the genome). No obvious differences in growth rates and sporulation of the strains were found. An artificially circularized S. coelicolor genome with deletions of total c. 1.6 Mb segments (840-kb for the left and 761-kb for the right arm of the linear chromosome) was obtained. The actinorhodin biosynthetic gene cluster could be overexpressed in some of the constructed strains.     

A gene encoding a homologue of dolichol phosphate-beta-D-mannose synthase is required for infection of Streptomyces coelicolor A3(2) by phage (phi)C31

We have shown previously that a gene encoding a homologue to the eukaryotic dolichol-phosphate-D-mannose, protein O-D-mannosyltransferase, was required for (phi)C31 infection of Streptomyces coelicolor. Here we show that a gene encoding the homologue to dolichol-phosphate-mannose synthase is also essential for phage sensitivity. These data confirm the role of glycosylation in the phage receptor for (phi)C31 in S. coelicolor.     

Cloning and expression of chitinase A gene from Streptomyces cyaneus SP-27: the enzyme participates in protoplast formation of Schizophyllum commune

Chitinase A of Streptomyces cyaneus SP-27 or chitinase I of Bacillus circulans KA-304 showed the protoplast-forming activity when combined with alpha-1,3-glucanase of B. circulans KA-304. The gene of chitinase A was cloned. It consisted of 903 nucleotides encoding 301 amino acid residues, including a putative signal peptide (35 amino acid residues). The deduced N-terminal moiety of chitinase A showed sequence homology with the chitin-binding domain of chitinase F from Streptomyces coelicolor and chitinase 30 from Streptomyces olivaceoviridisis. The C-terminal moiety also showed high sequence similarity to the catalytic domain of several Streptomyces family 19 chitinases as well as that of chitinase I of B. circulans KA-304. Recombinant chitinase A was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the recombinant enzyme were almost the same as those of chitinase A purified from a culture filtrate of S. cyaneus SP-27. The recombinant enzyme was superior to B. circulans KA-304 chitinase I not only in respect to protoplast forming activity in a mixture containing alpha-1,3-glucanase, but also to antifungal activity and powder chitin-hydrolyzing activity.     

Chitinase inhibitor allosamidin promotes chitinase production of Streptomyces generally

Allosamidin is a family 18 chitinase inhibitor produced by Streptomyces. In its producing strain, Streptomyces sp. AJ9463, allosamidin promotes production of the family 18 chitinase originated from chi65 in a chitin medium through the two-component regulatory system encoded by chi65R and chi65S, which were present at the 5'-upstream region of chi65. In this study, we showed generality of the allosamidin's effect. Allosamidin enhanced production of the family 18 chitinases originated from chi65h of Streptomyces halstedii MF425, another allosamidin producer, chiC of Streptomyces coelicolor A3(2) and chiIII of Streptomyces griseus. All the three chitinase genes had high homology to chi65 and two genes homologous to chi65S and chi65R were present at their 5'-upstream regions. When allosamidin's effect was tested with six Streptomyces strains randomly isolated from soil, allosamidin enhanced chitinase production of all strains. All six strains possessed a set of three genes homologous to chi65, chi65S and chi65R. Analysis of 16S rDNA indicated that allosamidin-sensitive strains are distributed widely in Streptomyces. These observations suggested that allosamidin can affect the common regulatory system for production of a chitinase with a two-component regulatory system in Streptomyces.