硫霉素生物合成酶基因克隆的研究

利用NTG诱变从硫霉素产生菌中获得了生物合成阻断变株Y₃。通过对Y3变株原生质体形成、再生条件及DNA转化的研究,初步建立了以变株为受体的克隆系统,以pIJ680为载体,从硫霉素产生菌S．Cattleya中鸟枪克隆,获得了能使Y₃变株恢复产生硫霉素的酶基因。根据对Y₃积累的中间产物的分析,认为该酶基因可能与硫霉素生物合成过程中肽的环化作用有关。重组质粒分子大小为9．8kb左右,插入片段大小为4．5kb,分子杂交试验证明插入片段来源于硫霉素产生菌S．Cattleya。     

螺旋霉素聚酮合成酶基因和抗性基因的克隆与表达的研究

根据不同聚酮合成酶基因DNA的同源性,利用放线紫红素聚酮合成酶基因act Ⅰ,actⅢ作探针,从螺旋霉索产生菌Str．spiramyceticus U-1941基因文库中检测并分离了螺旋霉素聚酮合成酶基因pCN3H8。限制酶酶切分析表明,其分子量为44kb。通过分子杂交实验,将螺旋霉素聚酮缩合酶基因(与act Ⅰ有同源性)及聚酮氧化还原酶基因(与actⅢ有同源性)进行了定位。pCN3H8 DNA在麦迪霉素产生菌变株Str.mycarofaciens sub sp．68中的表达产物,经紫外光谱分析与麦迪霉素相似。pCN3H8在放线紫红素聚酮缩合酶基因缺陷型变株Str．coelicolor TKl7中的表达产物,不具有放线紫红素的色素,其纸层析谱型与螺旋霉素有显著差别。pCN3H8在变青链霉菌Str．lividans TK24中的表达产物,也具有抗菌活性。将pCN3H8 DNA转化对螺旋霉素敏感的Str.griseofuscus原生质体,获得了螺旋霉素抗性的表达。从转化子中分离得到了质粒DNA pSG3,其分子量为7．0kb,可能是pCN3H8DNA转化Str．grlseofuscus时在体内缺失而形成。再转化实验证明,宿主菌对螺旋霉索的抗性,确实是由于pSG3 DNA作用的结果。含质粒pCG4,pSG3的螺旋霉素产生菌Str．Ambofaciens转化子螺旋霉素的产率明显提高。     

利用强启动功能片段提高麦迪霉素4”，-羟基丙酰化酶基因的表达

用PCR技术将本室克隆到的强启动功能片段取代麦迪霉素丙酰化酶基因(mpt)的启动子或与mpt基因自身启动子串连,获得含mpt重组质粒pCHFPE3和pCHFPE2。用含有这两个质粒的Streptomyces lividans TK24对螺旋霉素进行微生物转化,结果表明,与含有原启动子的mpt．S．Lividans TK24(p．WFPE)相比,丙酰螺旋霉素的组分比例分别提高了89．02％和58．53％。含重组质粒pCHFPE2的螺旋霉素产生菌S．Spiramyceticus发酵产物中丙酰螺旋霉素的组分也有较大辐度的提高。说明利用该强启动功能片段可以提高麦迪霉素丙酰化酶基因的表达。     

新型抗肿瘤力达霉素合成酶基因sgcD的克隆、表达和性质研究

力达霉素是由球孢链霉菌(Streptomyces globisporus)C1027产生的一种新型烯二炔类抗肿瘤抗生素, 具有很强的抗肿瘤活性. 从S. globisporus C1027中克隆获得长度为75 kb的力达霉素生物合成基因簇, 包含33个开放阅读框架(ORF). 以sgcD(ORF24)为研究对象, 对其功能进行了研究. 基因中断实验证明, sgcD与力达霉素生物合成相关. 经同源性分析, 推测sgcD编码氨基变位酶, 催化a-酪氨酸转化为b-酪氨酸, 参与力达霉素生物合成中β-酪氨酸的合成. 为了确定sgcD编码的酶的性质, 将sgcD克隆至大肠杆菌表达载体pET30a中进行诱导表达, 然后对表达产物进行酶学分析. 实验结果表明, sgcD的表达产物具有氨基变位酶的活性. sgcD编码的氨基变位酶是第一个被定性的烯二炔类抗肿瘤抗生素生物合成酶. 本研究将有助于这类抗生素生物合成机制的阐明, 对改造和研发新型抗肿瘤药物具有显著意义.     

麦迪霉素生物合成基因克隆研究

利用穿梭粘粒载体pNJ1组建了麦迪霉素产生菌Streptomyces mycarolaciens 1748的基因文库,用与麦迪霉素生物台成有类似途径的放线紫红素聚酮合成酶基因Act I,Act Ⅱ为探针,从麦迪霉素产生菌的基因文库中,获得了与ActI,Act Ⅱ基因有同源性的阳性克隆,对其中PCNSBl2、6C5及11E11克隆DNA进行了酶切分析,其分子量分别为36kb,48．5kb及41．6kb。PCN6C5 DNA中包含有PCN 8B32的全部DNA片段,PCN ⅡEⅡ与PCN 8B12及PCN 6C5有6．75kb的重叠区。通过分子杂交实验,初步将麦迪霉素聚酮台成酶基因定位在PCN 8812 DNA的EcoR I—BamH I 4．02kb片段上(与Act Ⅱ基因有同源性)及PCN 8B12(6C5),PCNIIEIIDNA BgⅡ—BgⅡ 2．42kb与PCNllE11 Bgl I—B g1 I 1Okb片段上(与Act I基因有同源性)。PCN 8B12及PCN 6C5克隆DNA在麦迪霉素聚酮合成酶基因缺陷型变株Streptomyces mycarofaciens var．68及不产抗生索的变铅青链霉菌Streptomyces lividans TK24受体菌的表达产物,经TLC及HPLC等分析表明与麦迪霉素标准品相似。     

黑曲霉pepD基因阻断突变菌株的构建及功能分析

运用同源重组技术破坏了黑曲霉基因组中的pepD基因,该基因编码一种类subtilisin的胞外蛋白酶PEPD。实验以黑曲霉GICC2773基因组DNA为模板,PCR扩增pepD基因,并在此基因中间插入潮霉素抗性基因(hph)表达单元,由此产生了3.7kb的pepD阻断基因片段。将此阻断基因片段与载体pBS连接,构建成pepD基因阻断质粒pBSDH。采用原生质体-CaCl₂/PEG法将酶切阻断质粒得到的含pepD基因和hph表达单元的3.7kb线性片段转化AspergillusnigerGICC2773菌株,在含潮霉素的平板上筛选潮霉素抗性转化子,从这些抗性转化子中经PCR检测分离到到1个pepD基因阻断突变菌株?pepD66。外源漆酶分泌活性分析显示,黑曲霉pepD基因的破坏使其外源漆酶的分泌表达有所提高。     

环状芽孢杆菌C-2几丁酶基因的克隆

环状芽孢杆菌(Bacillus circulans)C-2总DNA经PstI部分酶切后分离2～10kb的片段,插入质粒pUC19的PstI位点,转化大肠杆菌(Escherichia coli),利用几丁质平板从约8000个重组子中筛选到一个几丁酶基因阳性克隆(命名为pCHT1)。用12种限制酶对重组质粒进行的酶切分析表明,重组质粒中的插入片段长3.0kb,其中各有一个KpnI,SacI和SspI位点。把该克隆片段反向插入pUC19的PstI位点所得到的重组子同样具有几丁酶基因表达活性,说明此片段含有一个完整的几丁酶基因,其自身的启动子能被大肠杆菌转录系统所识别。Southern杂交证实了该片段来自于B.circulans C-2基因组,且以单拷贝形式存在,它不能与来自于其它7株几丁酶产生菌的总DNA杂交。     

苏云金杆菌δ—内毒素基因在大肠杆菌中的克隆及表达

分离了苏云金杆菌肯尼亚亚种7404(Bacillus huringiensis subsp．Kenyae 7404)和库斯塔克亚种(Bacillus thuringiensis subsp．Kwrstaki HD-1)的质粒。经凝胶原位杂交证明苏云金杆菌肯尼亚亚种7404的δ—内毒素基因位于约47Md大小的一个质粒上。用蔗糖密度梯度离心法从sau 3 A1部分酶解的上述两种苏云金杆菌质粒DNA酶解片段中分离出大于 4kb的DNA片段,将这些片段克隆到pBR332的Barn HI位点上并转化大肠杆菌HB101。通过菌落原位杂交、菌落原位放射免疫试验及Western blct分析等方法,选到了带有δ—内毒素基因并能在大肠杆菌中表达此毒蛋白的转化体。初步的生物学试验表明,在四个试验过的转化体中带有肯尼亚亚种δ—内毒素基因的转化体TK89及带有库斯塔克亚种δ—内毒素基因的转化体THl2和TH48对烟青虫(Heliothis assulta)有毒杀活性。     

琥珀酸弧菌L-天门冬酰胺酶基因的初级克隆和表达

本文以表达型噬菌体λgtll为载体,以及¹²⁵I标记的放射免疫抗体为探针,从EcDR I酶切的琥珀酸弧菌(Vyibrto succinogenes)染色体DNA片段中克隆得到携带天门冬酰胺酶基因的目的片段,在宿主菌E．Coil Y 1090 中得到表达。经酶解和凝胶电泳分析表明该插入DNA片段的分子量为5．8kb．重组DNA感染另一宿主菌E．ColiYl089后所产生的酶蛋白具有L-天门冬酰胺酶活力。用重组DNA(λgt11-AS8)为探针进行southern DNA杂交,琥珀酸弧菌染色体DNA的Ec0R I酶切片段中,出现一条位置在5．8kb处的杂交带,证明我们克隆到的携带L-天门冬酰胺酶基因的目的片段来自琥珀酸弧菌。     

棉铃虫颗粒体病毒增效蛋白基因2.1kb片段在大肠杆菌中的表达

以棉铃虫颗粒体病毒(Helicoverpa armigera granulosis virus,简称HaGV)基因组DNA为模板,设计引物PCR扩增病毒增效蛋白(Enhancin)基因,然后经SacI/PstI双酶切消化,得到5′端截短的约2.1kb增效蛋白基因片段,再与pQE30质粒连接,构建了重组表达载体pQE/EnC,转化大肠杆菌M15(pREP4),在IPTG诱导下表达出分子量约为78×10³ D的融合蛋白并命名为P78,纯化的P78包涵体显示了明显的增效活性,可提高AcMNPV对小菜蛾幼虫的感染率27.88%～32.92%。     

Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase from Streptomyces cacaoi

A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine beta-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces lividans TK24 using the high-copy-number plasmid pIJ702 as vector. A 30-fold higher yield of beta-lactamase was obtained from S. lividans strain ML1, carrying the recombinant plasmid pDML51, than from S. cacaoi grown under optimal production conditions. In all respects (molecular mass, isoelectric point, kinetics of inhibition by beta-iodopenicillanate) the overproduced S. lividans ML1 beta-lactamase was identical to the original S. cacaoi enzyme. A considerable reduction of beta-lactamase production was caused by elimination of a 12.8 kb portion of the 19 kb DNA fragment by cleavage at an internal SphI site located more than 3 kb upstream of the beta-lactamase structural gene. The beta-lactamase gene was located within a 1.8 NcoI-BclI fragment but when this fragment was cloned in S. lividans pIJ702, the resulting strain produced hardly any more beta-lactamase than the original S. cacaoi.     

Cloning and expression of a lignin peroxidase gene from Streptomyces viridosporus in Streptomyces lividans.

A lignin peroxidase gene was cloned from Streptomyces viridosporus T7A into Streptomyces lividans TK64 in plasmid pIJ702. BglII-digested genomic DNA (4-10 kb) of S. viridosporus was shotgun-cloned into S. lividans after insertion into the melanin (mel+) gene of pIJ702. Transformants expressing pIJ702 with insert DNA were selected based upon the appearance of thiostrepton resistant (tsrr)/mel-colonies on regeneration medium. Lignin peroxidase-expressing clones were isolated from this population by screening of transformants on a tsr-poly B-411 dye agar medium. In the presence of H2O2 excreted by S. lividans, colonies of lignin peroxidase-expressing clones decolorized the dye. Among 1000 transformants screened, 2 dye-decolorizing clones were found. One, pIJ702/TK64.1 (TK64.1), was further characterized. TK64.1 expressed significant extracellular 2,4-dichlorophenol (2.4-DCP) peroxidase activity (= assay for S. viridosporus lignin peroxidase). Under the cultural conditions employed, plasmidless S. lividans TK64 had a low background level of 2.4-DCP oxidizing activity. TK64.1 excreted an extracellular peroxidase not observed in S. lividans TK64, but similar to S. viridosporus lignin peroxidase ALip-P3, as shown by activity stain assays on nondenaturing polyacrylamide gels. The gene was located on a 4 kb fragment of S. viridosporus genomic DNA. When peroxidase-encoding plasmid, pIJ702.LP, was purified and used to transform three different S. lividans strains (TK64, TK23, TK24), all transformants tested decolorized poly B-411. When grown on lignocellulose in solid state processes, genetically engineered S. lividans TK64.1 degraded the lignocellulose slightly better than did S. lividans TK64. This is the first report of the cloning of a bacterial gene coding for a lignin-degrading enzyme.     

头状轮生链霉菌中丝裂霉素C抗性基因的克隆及功能研究

头状轮生链霉菌(\%Streptoverticillium caespitosus\%)ATCC27422是抗肿瘤药物丝裂霉素的主要产生菌,实验通过诱变筛选获得不产生丝裂霉素同时对丝裂霉素C敏感的阻断变种S6,并以它为受体宿主,以质粒pIJ699为载体,建立野生型头状轮生链霉菌菌株ATCC27422的基因库。采用鸟枪法克隆技术,从库中筛选获得含有丝裂霉素C抗性基因的62kb外源片段的克隆子。将含此外源片段的质粒pLX5导入变铅青链霉菌(\%Streptomyces lividans\%)获得表达。并且首次成功地运用电穿孔法将pLX5导入野生型菌株中,使其对丝裂霉素C的抗性大幅度提高：最低抑制浓度(MIC)由原来的200μg/mL上升至1000μg/mL以上。摇瓶发酵实验表明：单位菌量的ATCC27422(pLX5)的丝裂霉素产量高于野生菌株ATCC27422,因此丝裂霉素C抗性与产量之间存在一定的相关性。     

Identification, isolation and sequencing of the recA gene of Streptomyces lividans TK24

Abstract An internal fragment of the recA gene of Streptomyces cattleya was amplified by the polymerase chain reaction (PCR) employing degenerate oligonucleotide primers. Using this fragment as a hybridization probe, a recA homologous gene could be shown in each tested Streptomyces strain. A 4.4 kb Bam HI fragment which carried the complete recA gene was isolated from Streptomyces lividans TK24. Sequence analysis suggested that the coding region of the recA gene consists of 1122 bp. The highest similarity (∼78%) could be detected to the recA genes of Mycobacterium tuberculosis and Mycobacterium leprae . After fusion with an E. coli promoter the S. lividans recA gene could partially complement an Escherichia coli recA mutant.     

Molecular cloning and sequencing of a non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762.

A bromoperoxidase gene (bpoT), recently cloned from Streptomyces aureofaciens Tü24, was used as a probe in Southern blot hybridization of total DNA from S. aureofaciens ATCC 10762. A single SstI fragment of 5.4 kb was detected, which was cloned via an enriched gene library into Escherichia coli. The functional bromoperoxidase gene was located on a 2.1 kb BamHI-HindIII fragment by subcloning into S. lividans TK64, using the multicopy plasmid pIJ486. The enzyme was overproduced in S. lividans TK64 (up to 30,000 times compared to S. aureofaciens ATCC 10762) and showed the same electrophoretic and immunological properties as the bromoperoxidase BPO-A2 purified from S. aureofaciens ATCC 10762. DNA sequence analysis revealed an open reading frame encoding a predicted polypeptide with the same M(r) and N-terminal amino acid sequence as the purified subunit of BPO-A2.     

Cloning and heterologous expression of the entire set of structural genes for nikkomycin synthesis from Streptomyces tendae Tü901 in Streptomyces lividans.

A genomic library from Streptomyces tendae raised in shuttle cosmid vector pKC505 was screened with a previously isolated 8-kb DNA fragment containing the orfP1 gene, which is involved in nikkomycin biosynthesis. The entire set of structural genes for nikkomycin synthesis was heterologously expressed in S. lividans TK23 by introducing recombinant cosmids p24/32 and p9/43-2, carrying inserts of about 31 and 27 kb, respectively, overlapping by 15 kb. S. lividans transformants synthesized nikkomycins X, Z, I, and J, which were identified by high-pressure liquid chromatography analyses of culture filtrates.     

Cloning of a thermostable alpha-amylase gene from Thermomonospora curvata and its expression in Streptomyces lividans

The gene from Thermomonospora curvata CCM 3312 coding for thermostable alpha-amylase (tam) has been cloned in Streptomyces lividans TK 24 and localized to a 2.6 kb HindIII-BamHI fragment of DNA. The data presented here show that the tam gene is expressed at a high level in S. lividans and that the protein is efficiently excreted.     

苏云金芽孢杆菌cry1基因在荧光假单胞菌Pfx-18中的表达特性

用DIG标记的cry1Aa基因EcoRI-F片段的RNA探针,对筛选的鳞翅目高毒力菌株的质粒进行Southern分析,将cry基因定位在39．3MD质粒上。该质粒经HindⅢ酶解,用同样探针进行杂交,呈现6．5kb和7．1kb两条阳性带,其中7．1kb片段的杂交强度明显高于6．5kb片段。将7．1kb片段与多寄主质粒pSUP106连接,转化荧光假单胞菌Pfx－18,获得克隆子LZP-1。克隆基因用PCR鉴定,显示典型的cry1Ab谱带。经SDS-PAGE分析,克隆株表达66kD杀虫晶体蛋白和一些小分子多肽。其发酵液稀释1000倍对三龄小菜蛾幼虫的致死率为33％。     

毒性T淋巴细胞相关抗原-4(CTLA-4)在链霉菌中的表达研究

应用两种链霉菌新型信号肽--vsi和gpp在常用工程菌变铅青链霉菌（Streptomyces lividans）中进行了CTLA-4的分泌表达研究,vsi信号肽与CTLA-4的融合片段克隆至链霉素-大肠杆菌穿梭质粒pUWL-219,同时gpp信号肽与CTLA-4片段在质粒pLNSP中融合,分别转化S.lividans TK24,获得重组菌株S.lividans[pUWL219-VC]和S.lividans[pLNSP/CTLA-4]。重组菌株的发酵上清液经SDS-PAGE及Western blotting分析结果表明：应用不同信号肽构建的两株工程菌均能表达分子量为13000重组蛋白,具有免疫活性。     

Cloning and expression of a nitroaryl reductase gene from Streptomyces aminophilus strain MCMB411 in E. coli JM109 and Streptomyces lividans TK64

A partial genomic library was prepared in E. coli JM109 using pBR322 as vector and 2.4 kb Sau 3A I chromosomal fragment, encoding a nitroaryl reductase (nbr A) gene, from Streptomyces aminophilus strain MCMB 411. From the library, 2.4 kb fragment was recloned in E. coli JM109 and S. lividans TK64 using pUC18 and pIJ702 as vectors respectively. The recombinant plasmids pSD103 and pSD105 expressed the reductase gene and exported the enzyme in periplasmic space of E. coli and in cytoplasm of S. lividans TK64. The proteins expressed by E. coli and S. lividans had the same molecular mass (70 kD) as that expressed by parent strain, which suggested that the enzyme was processed similarly by all strains. Activities of the enzymes cloned in E. coli JM109 and S. lividans TK64 containing recombinant plasmids pSD103 and pSD105 respectively were optimum at 30 degrees C and pH 9 and requirement of cofactors was same as that of the parent strain.