黑暗链霉菌安普霉素生物合成基因aprG的克隆和功能研究

从安普霉素产生菌S．tenebrarius H6总DNA中已克隆得到8．2kb的安普霉素抗性基因连锁片段。对距离抗性基因下游4．5kb的SacI-Sau3AI片段进行测序分析，发现了一个新的891bp开放阅读框架。推测其编码为296个氨基酸的蛋白质。经BLASTX程序分析，没有发现与之同源的基因，是一个新基因，该基因命名为aprG。构建基因阻断穿梭载体pSPU58，经接合转移导入S．tenebrarius H6，筛选单交换阻断变株，并用Southern blot验证阻断变株的aprG已经被破坏。经发酵分析，阻断变株不再合成安普霉素，表明aprG与安普霉素生物合成直接相关。     

Geldanamycin产生菌生物合成相关基因的克隆与分析

目的：从geldanamycin产生菌吸水链霉菌（S.hygroscopicus)中克隆生物合成基因，为阐明生物合成机制及改造其结构奠定基础。方法：以链霉菌／大肠埃希氏菌穿梭cosmids pKC505为载体构建了插入片段为20－30kb的S.hygroscopicus总DNA文库，以利福霉素中与3－氨基－5－羟基苯甲酸（AHBA）合成相关基因为探针，经菌落杂交，Southern杂交筛选同源基因片段，测序结果与Genbank进行同源性比较分析，并以attp-噬菌体载体KC515利用基因阻断实验对克隆的基因片段进行功能鉴定。结果：构建的基因文库含重组基因比率高，基因组覆盖率高，稳定性好，经同源基因探针杂交，从文库中筛选出9个阳性cosmids克隆pCGB20,pCGB26,pCGB28,pCGB29,pCGB36,pCGB45,pCGB49,pCGB63,pCGB75，测序分析表明，cosmid pCGB20中BamHI-BamHI 8kb片段两端的不完整ORF编码蛋白与竹桃霉素（oleandomycin)PKS Module 5,Module 6(一致性为79％）和红霉内酯合成酶ORF2（一致性为75％），ORF1（一致性为73％），ORF3（一致性为70％），高度同源；BamHI-BamHI 3kb片段一端与编码类结核分枝杆菌的磷酸吡哆醛依赖的氨基转移酶家族中半胱氨酸脱硫酶的DNA有同源性，该家族与AHBA合成酶关系密切，Cosmid pCGB20的BamHIBamHI 8kb及BamHI-BamHI 3kb基因片段已通过基因阻断实验初步鉴定，证明它们参与geldanamycin生物合成，而其他cosmids阳性克隆的序列分析及功能研究尚在进行中。     

ClyA基因的克隆及检验

用PCR法从大肠杆菌中调出长1.9kb的DNA片断，插入质粒pT7-7，核酸电泳和DNA测序分析结果证明该片段是GlyA目的基因。     

汉坦病毒M和S片段基因的克隆及在成纤维细胞中的瞬时表达

目的构建汉城病毒M片段和汉滩病毒部分S片段的重组基因pEGFP-M-S，并在成纤维细胞L929中进行瞬时表达。方法用PCR方法克隆分别装在两个模板载体中的部分M片段，连接人pcDNA3．1载体，利用简并密码子得到完整的M片段。将汉城病毒M片段开放阅读框全长3．4kb的基因与装有汉滩病毒S片段5’末端0．7kb基因的载体pEGFP-HTNV-S0．7用酶切位点连接，构成4．1kb的重组基因，并转染成纤维细胞，进行48h、72h表达。结果成功得到预期大小的4．1kb的重组基因，并在成纤维细胞中进行了瞬时表达。结论汉城病毒M片段和汉滩病毒部分S片段基因的重组基因在成纤维细胞中瞬时表达，产生了M片段的约70kD和55kD的两个糖蛋白，并在与S片段拼接处经蛋白酶切割产生了约26kD的核蛋白，免疫印迹分析产生的约26kD的蛋白具有抗汉坦病毒的抗原活性。     

快速定量液体杂交法检测血清中HBV DNA

这种方法的原理是夹心杂交探针。乙型肝炎病毒(HBV)ayw亚型DNA经Bam HI酶切割成两段:1.5千碱基对(kb)片段含有核心抗原基因,1.7kb片段含有表面抗原基因。前者克隆于噬菌体M13mp 10和11,并经生物素化化学处理作为捕获探针,后者克隆于pBR322,经~35S三磷酸脱氧胞苷(dCTP)标记     

Streptomyces sp.4353中3-氨基-5-羟基-苯甲酸（AHBA）合酶基因的克隆及其在大肠埃希菌中的表达

目的 克隆Streptomyces sp.4353中的3-氨基-5-羟基-苯甲酸(AHBA)合酶基因,并将其在大肠埃希菌中表达.方法参与AHBA生物合成的5个基因,即氨基奎尼酸脱水酶基因、氨基奎尼酸/莽草酸脱氢酶基因、AHBA合酶基因、氧化还原酶基因和磷酸酶基因通常连锁,形成一个AHBA生物合成基因簇.本文通过分析、比较已知AHBA生物合成基因簇的基因组织结构特点和序列保守性,设计引物,以Streptomyces sp.4353的总DNA为模板,在已知737bp AHBA合酶基因部分序列的基础上,通过PCR扩增其上、下游DNA序列,克隆至pMD18-T载体上,测序、拼接并进行生物信息学分析.以pET24a(+)为表达载体,对克隆得到的AHBA合酶基因在大肠埃希菌BL21-Codon Plus(DE3)-RIL中进行表达和SDS-PAGE检测.结果从Streptomyces sp.4353中得到了一段2872bp大小的DNA片段(NCBI GenBank接受号EU734184),其中含有AHBA生物合成基因簇中的AHBA合酶基因、氧化还原酶基因和磷酸酶基因(部分);AHBA合酶基因在大肠埃希菌中得到了成功表达,在SDS-PAGE上可见到清晰的、与预计大小(42.7ku)一致的蛋白表达特异条带.结论 Streptomyces sp.4353中的AHBA合酶基因的克隆以及在大肠埃希菌中的表达为进一步研究该基因的功能奠定了基础.本研究克隆得到的2872bp DNA片段还有可能用于AHBA的杂合/异源生物合成,以及安莎类抗生素的组合生物合成.     

含Butirosin产生菌环状芽孢杆菌NRRL-B3312启动子DNA片段的No.44重组质粒的构建及其性质

利用启动基因探针质粒pPL603作为载体,将Butirosin产生菌环状芽孢杆菌NRRL-B3312启动子DNA片段插入到ppL603的EcoRI位点,获得了分子量为8.48kb,氯霉素抗性水平比pPL603提高16倍的No.44重组质粒(50μg/ml).建立了No.44重组质粒的酶切图谱,Sall、Bgll、HindⅢ、Aval和SmallNo.对44重组质粒的酶切位点数目分别为1、2、3、3和3个,而KpnⅠ、XhoⅠ、SstⅠ和SstⅡ对No.44重组质粒没有切口.经缺失重组实验初步确定了No.44重组质粒插入片段的启动子功能区域,启动区域应在0.38kb EcoRⅠ-AvaⅠ间的DNA片段和1.21kb AvaⅠ-EcoRⅠ间的DNA片段两部分之一中.     

同源重组方法在制备DNA大片段中的应用

目的介绍快速、简便制备DNA大片段的新方法。方法查阅国内外近年关于DNA大片段制备方法的文献33篇,对这些方法进行归纳和总结。结果综述了大肠杆菌Red/ET重组系统和基于酿酒酵母重组系统的DNA自组装和TAR克隆在DNA片段制备中的应用。结论同源重组系统不仅提高了克隆DNA大片段的效率,而且简化了操作流程。这些方法在制备DNA大片段用于组合生物合成和合成生物学制备药物中具有重要的应用前景。     

真菌Drechslera catenaria聚酮类代谢产物及聚酮合成酶基因的初步研究

目的 分离分析内脐蠕孢真菌(Drchslera catenaria)的次级聚酮类代谢产物,初步分析其非还原型Ⅰ型聚酮合成酶基因组成,为该菌株中大黄酚生物合成机制的阐明奠定理论基础.方法 应用Czapek Dox培养基发酵培养,酸化乙酸乙酯提取,硅胶柱层析及薄层制备等方法,对该菌株产生的聚酮类代谢产物进行分离纯化;利用紫外(UV),液相-质谱联用(LC-MS)和核磁共振谱(1H-NMR)鉴定化学结构;基于真菌聚酮合成酶保守序列设计引物,克隆与代谢产物合成相关的聚酮合成酶基因.结果与结论 从试验菌株菌丝体及发酵液中分离获得4个芳香聚酮类化合物,其结构分别为大黄酚(chrysophanol),长蠕孢素(helminthosporin),冰岛青霉素(islandicin),链蠕孢素(catenarin),其中后3个为首次从该真菌中得到.从基因组DNA中得到一段非还原性聚酮合成酶(PKS)基因序列(约5.3kb),与Pyrenophora tritici-repentis Pt-1C-BFP中黄色色素合成相关的基因片段有较高类似度,很有可能与内脐蠕孢真菌中大黄酚的生物合成有关;在距离该基因2.5kb处克隆到一段基因序列(0.79kb),与P.triticirepentis Pt-1C-BFP中β-酮酯酰基-ACP-还原酶(KR)基因完全相似,其很可能负责大黄素的6位羰基经还原形成大黄酚.     

用PCR技术从cDNA文库中获取目的基因长片段

目的　建立一种简便有效的获取cDNA大片段的实验技术。方法　应用PCR原理 ,设计特异引物结合文库载体引物对大鼠胎肝cDNA文库进行PCR扩增 ;用随机引物标记法标记已知小片段“目的基因”(30 0bp± )作为探针 ,对PCR产物进行Sorthern杂交以检验“目的基因”片段的正确性及长度 ;回收“目的基因”片段并连接入PGEM T载体 ,转染JM 10 9并扩增后 ,提取“目的基因”测序。结果　琼脂糖凝胶电泳发现PCR扩增产物有多条不同长度的段 ,Sorthern杂交发现一段长度为 1 5kb的cDNA片段为“目的基因” ;测序结果与GeneBank进行Blaste分析 ,该基因为一株新的序列 ,已在GeneBank中登录注册 (RatLiverRegeneration relatedPro tein 1,RLRP 1AF2 36 0 5 4 )。结论　应用PCR结合Sorthern杂交等手段可迅速从cDNA文库中获取长片段目的基因     

The leptomycin gene cluster and its heterologous expression in Streptomyces lividans

Leptomycin exerts its antifungal and anti-tumoral activity via inhibiting nucleo-cytoplasmic translocations in eukaryotic cells. To learn more about the biosynthesis of leptomycin and in an effort to generate leptomycin analogues through genetic engineering, 90 kb segment of DNA containing the putative leptomycin (lep) biosynthesis cluster from Streptomyces sp. ATCC 39366 was cloned and sequenced. The lep cluster consist of 12 polyketide synthase (PKS) modules distributed in four genes (lepA, B, C and D) and a P450 encoding gene. The lep gene cluster was confirmed by its successful expression in Streptomyces lividans, where it directed the production of the two natural congeners-leptomycins A and B. The production of leptomycin B showed that the host has the capability to synthesize ethylmalonyl-CoA.     

Cloning of large DNA fragments, which hybridize with actinorhodin biosynthesis genes, from kalafungin and nanaomycin A methyl ester producers and identification of genes for kalafungin biosynthesis of the kalafungin producer

Large actI, III-homologous DNA fragments were isolated from genomic libraries of the strains that produce the benzoisochromanequinone antibiotics kalafungin and nanaomycin A methyl ester, Streptomyces tanashiensis strain Kala and Streptomyces sp. OM-173, respectively. These libraries were prepared in Escherichia coli JM108 by using a novel Streptomyces-E. coli bifunctional cosmid, pKU205, and screened with polyketide synthase genes (actI and III) for actinorhodin biosynthesis from Streptomyces coelicolor A3(2) as probes. The cloned DNA fragments (28 and 42 kb) were analyzed by hybridization with DNA containing actinorhodin biosynthetic genes (actI, II, III, IV, VA, VB, VI and VII). Both fragments hybridized with the actI, III, VA and VI regions, but not with the actII, IV, VB and VII regions. The cloned fragment of S. tanashiensis DNA was analyzed by complementation tests with kalafungin-nonproducing mutants. Seven genes (kalI approximately VII), which correspond to seven steps in kalafungin biosynthesis, were found to be located on a 14 kb continuous DNA fragment. Five of the genes were located on the regions homologous to the genes for actinorhodin biosynthesis, but the other two genes were not. Although kalafungin is an intermediate or shunt product in actinorhodin biosynthesis in S. coelicolor A3(2), the genes for kalafungin biosynthesis in S. tanashiensis are not identical with those in S. coelicolor A3(2).     

Cloning of aklavinone biosynthesis genes from Streptomyces galilaeus.

Aklavinone is an aglycone of aclacinomycin A which is an important antitumor drug. Genes for the biosynthesis of aklavinone were cloned from Streptomyces galilaeus 3AR-33, an aklavinone-producing mutant, by use of the actI and actIII polyketide synthase gene probes. Restriction mapping and Southern analysis of the DNA cloned in a lambda phage vector established that the DNA represented three different regions of the S. galilaeus 3AR-33 genome that contained 3.4, 2.5, and 4.1 kb BamHI fragments which hybridized with actIII. Of those, only the 3.4 kb fragment also hybridized with actI. Complementation experiments with specifically blocked mutants confirmed that the cloned 3.4 kb BamHI fragment contains the genes required for the early stage of polyketide synthesis in aklavinone biosynthesis.     

Phenotypic changes associated with loss of expression of tylosin biosynthesis and resistance genes in Streptomyces fradiae

Two mutants of the tylosin-producing Streptomyces fradiae defective in the biosynthesis of the macrolide antibiotic tylosin were isolated from colonies derived from regenerated protoplasts. Both strains were unable to carry out any of at least seven tylosin biosynthetic steps and were sensitive to tylosin. One strain, JS82, was also more sensitive to chloramphenicol (Cm), mitomycin C (Mc), hygromycin B (Hm) and kanamycin (Km) than its parent strain. The other strain, JS87, was also more sensitive to Cm than wild type but expressed normal levels of resistance to Mc and Hm. Both strains expressed genetic instabilities associated with auxotrophy or expression of antibiotic resistance. Since the genetic instabilities were not due to defective error-free or error-prone DNA repair, they appear to be due to genetic rearrangements associated with the deletion or amplification of sequences linked to and perhaps encompassing tylosin biosynthesis genes.     

棒状链霉菌中lat基因的置换与克拉维酸的选择性生产

目的提高棒状链霉菌的克拉维酸生产能力 ,去除副产物头霉素C。方法将安普霉素抗性基因片段插入到 4 7kb的lat pcbAB基因片段中 ,采用接合转移方法对棒状链霉菌中头霉素C生物合成的关键基因lat基因进行了置换。结果与结论经PCR验证 ,染色体上正常的lat pcbAB基因被插入失活的基因所替代。对重组菌株的发酵产物进行HPLC检测 ,发现所有的重组菌株均不再合成头霉素C ,并且重组菌株C35 85 lat ::apr、C2 5 1 lat::apr和C16 6 lat::apr的克拉维酸产量较出发菌株分别提高了 2 6 7 6 8%、4 3 6 9%和 35 0 5 %。结果表明基因插入失活是去除多余组分 ,提高目标组分产量的可行途径     

Partial activation of a silent angucycline-type gene cluster from a rubromycin beta producing Streptomyces sp. PGA64

In the course of DNA-fingerprinting our strain collection for antibiotic biosynthesis genes, two different type II polyketide synthase (PKS) gene clusters were observed from Streptomyces sp. PGA64. Phylogenetic analysis placed these together with known rubromycin and angucycline biosynthetic gene clusters. The host strain itself has a very clean production profile of secondary metabolites, which composes mainly of rubromycin beta under typical fermentation conditions. Sequencing of a 16.5 kb fragment from the putative angucycline cluster revealed eight genes that were homologous to typical type II PKS genes responsible for synthesizing aromatic polyketides. These genes were especially similar to genes from known angucycline biosynthetic gene clusters and also synteny to these clusters was observed. In addition, three genes were recognized that are needed for priming the minimal PKS complex before polyketide synthesis can initiate, but which are not normally found to cluster with antibiotic biosynthesis genes. A putative repressor gene that was dissimilar to repressor genes found from well-characterized antibiotic biosynthesis gene clusters was also discovered. Gene disruption of the repressor resulted in partial activation of the cluster and production of two angucycline metabolites, UWM6 and rabelomycin. The results confirm that the DNA-fingerprinting method we have developed can be used to correctly detect compounds that are not visible in chemical screens.     

The complete biosynthetic gene cluster of the 28-membered polyketide macrolactones, halstoctacosanolides, from Streptomyces halstedii HC34

Halstoctacoanolides A and B are 28-membered polyketide macrolactones and were isolated from Streptomyces halstedii HC34. The biosynthetic gene cluster (hls cluster) of halstoctacosanolides was completely identified from the genome library of Streptomyces halstedii HC34. DNA sequence analysis of ca. 100 kb region revealed that there were seven type I polyketide synthases (PKSs) and two cytochrome P450 monooxygenases in this cluster. Involvement of the gene cluster in the halstoctacosanolide biosynthesis was demonstrated by the gene disruption of P450 monooxygenase genes. The mutants produced a new deoxygenated halstoctacosanolide derivative, halstoctacosanolide C, which confirmed that the hls gene cluster was essential for the biosynthesis of halstoctacosanolides.     

Molecular cloning of a xylanase gene from Streptomyces sp. No. 36a and its expression in streptomycetes

The gene for an extracellular xylanase from Streptomyces sp. No. 36a was cloned into Streptomyces lividans TK21 using pIJ702 as a vector plasmid. The smallest DNA fragment encoding the xylanase gene and its possible promotor was determined to be a 1.04 kb Sph I-Sac I fragment by sub-cloning studies. This xylanase gene fragment was transferred into the pSK2 series of plasmids and introduced into Streptomyces kasugaensis G3 protoplasts. The cloned xylanase gene was expressed in both S. lividans TK21 and S. kasugaensis G3, and these clones produced and secreted high yields of xylanase into the culture medium. The xylanase production was not detected when a foreign DNA fragment was inserted into the Bcl I site locating in the xylanase gene fragment.     

kasT gene of Streptomyces kasugaensis M338-M1 encodes a DNA-binding protein which binds to intergenic region of kasU-kasJ in the kasugamycin biosynthesis gene cluster

We previously reported that a 4.2 kb SacI-EcoRI DNA region from Streptomyces kasugaensis M338-M1, a kasugamycin (KSM) producer, included KSM transporter genes (kasKLM). As an extension of that study, a 3.7 kb Psti-SacI DNA region, located at 1.5 approximately 5.2 kb upstream of kasK, was cloned and sequenced, revealing three complete open reading frames, designated kasT, kasU and kasJ. The kasJ gene encodes a protein (KasJ) with a conserved dinucleotide (FAD)-binding motif Homology search for KasJ showed its similarity to NADH: N-amidino-scyllo-inosamine oxidoreductase (StsB) which is involved in biosynthesis of the streptidine moiety of streptomycin (SM) in S. griseus. The kasT gene encodes a DNA-binding protein (KasT), including a helix-turn-helix motif near the center of the sequence. This protein is similar in structure to a pathway-specific activator protein (StrR) that plays a role in regulating the SM biosynthesis gene cluster of S. griseus. A fusion protein (Trx-KasT) clearly showed DNA binding activity with the intergenic region of kasU-kasJ, suggesting that KasT is a pathway-specific regulator of the KSM biosynthesis gene cluster.