链霉菌基因转移的方法

本文介绍了用于链霉菌基因转移的各种方法 ,涉及原生质体转化、电穿孔、接合转移和噬菌体转导 ,对影响链霉菌基因转移的各种因素进行了分析。     

Biosynthesis regulation of natamycin production from Streptomyces natalensis HDMNTE-01 enhanced by response surface methodology

Natamycin has been widely applied in medical treatments and food protection widely due to its effective inhibition to the growth of yeast and mold. As polyene macrolide antibiotic, the biosynthesis pathway of natamycin is relatively clear. To regulate the biosynthesis of natamycin, additions of precursors affecting cell growth and natamycin production were investigated. The results showed that 0.003% (w/v) potassium ferrocyanide and sodium propionate: n-butanol at a ratio of 4:1 was added into the broth at 0 and 24?hr, respectively, and they contributed to yield natamycin, reaching 1.62?g?L^?1 (174.6% higher than control). Furthermore, response surface methodology was undertaken to enhance natamycin production by Streptomyces natalensis HDMNTE-01 (a wild strain). The optimum conditions determined were: glucose 3.97%; soya peptone 2%; yeast extract 0.5%; original pH 7.0; inoculum volume 6%; growth in a 250-mL flask containing 24.68?mL of medium; shaken (220?rpm) at 28°C for 4 days. Under the optimized conditions, the yield was 2.81?g?L^?1 natamycin in 5-L fermentor when the fermentation was processed.     

Fostriecin产生菌Streptomyces pulveraceus遗传转化体系的建立与优化

【目的】建立并优化链霉菌Fostriecin产生菌Streptomyces pulveraceus的遗传转化系统。【方法】以整合型质粒pSET152为出发质粒,通过供体菌E.coli ET12567/pUZ8002与受体菌Streptomyces pulveraceus进行接合转移。【结果】确定了链霉菌Streptomyces pulveraceus的最佳接合转移条件:培养基为终浓度含15%甘氨酸的MS培养基;孢子热激条件为50°C 10 min;阿伯拉霉素覆盖的时间为18 h,终浓度为20 mg/L。同时,把组成型启动子ermE+与绿色荧光蛋白基因(gfp)克隆到pSET152载体上,通过接合转移整合到该链霉菌中,gfp获得表达。【结论】建立Fostriecin产生菌的遗传转化系统,并发现甘氨酸能显著提高链霉菌的接合转移效率。     

海洋来源链霉菌OUCMDZ-3434中wblA基因的功能

【背景】Streptomyces sp. OUCMDZ-3434分离自山东青岛栈桥浒苔样品,其次级代谢产物Wailupemycin类化合物具有良好的α-糖苷酶抑制活性,但产量很低,影响了其进一步研发。【目的】建立海洋来源链霉菌Streptomyces sp. OUCMDZ-3434菌株的遗传转化系统,通过阻断全局性调控基因wblA探究Wailupemycin类化合物产量变化。【方法】以p SET152为载体,采用大肠杆菌-链霉菌属间接合转移策略建立了Streptomycessp.OUCMDZ-3434的遗传转化方法。采用PCR-Targeting策略构建了wblA基因阻断突变株,通过HPLC分析野生型和突变株发酵产物的差异并对表型差异进行显微形态观察。【结果】建立了Streptomyces sp. OUCMDZ-3434遗传转化系统,构建了wblA基因阻断突变株,与野生株相比,突变株发酵产物中Wailupemycin G产量提高了3倍,同时丧失了产生孢子的能力。【结论】在Streptomycessp.OUCMDZ-3434中wblA对Wailupemycin类化合物生物合成起到了负调控作用,同时参与调控孢子形成,本研究为采用遗传改造策略提高Wailupemycin G产量提供了参考。     

Use of a cloned gene involved in candicidin production to discover new polyene producer Streptomyces strains

A p-aminobenzoic synthase gene (pabS) from Streptomyces griseus IMRU 3570 involved in candicidin production was used as probe to find new aromatic polyene producing Streptomyces strains. The pab gene hybridizes with 6 out of 16 Streptomyces strains, and those strains which hybridize turned out to be polyene producers. Such strains were never before described as polyene producers.     

The two-component phoR-phoP system of Streptomyces natalensis: Inactivation or deletion of phoP reduces the negative phosphate regulation of pimaricin biosynthesis

The biosynthesis of the antifungal pimaricin in Streptomyces natalensis is very sensitive to phosphate regulation. Concentrations of inorganic phosphate above 1mM drastically reduced pimaricin production. At 10mM phosphate, expression of all the pimaricin biosynthesis (pim) genes including the pathway-specific positive regulator pimR is fully repressed. The phoU-phoR-phoP cluster of S. natalensis encoding two-component Pho system was cloned and sequenced. Binding of the response regulator PhoP to the consensus PHO boxes in the phoU-phoRP intergenic promoter region was observed. A phoP-disrupted mutant and a phoR-phoP deletion mutant were obtained. Production of pimaricin in these two mutants increased up to 80% in complex yeast extract-malt extract (YEME) or NBG media and showed reduced sensitivity to phosphate control. Four of the pim genes, pimS1, pimS4, pimC and pimG showed increased expression in the phoP-disrupted mutant. However, no consensus PHO boxes were found in the promoter regions of any of the pim genes, suggesting that phosphate control of these genes is mediated indirectly by PhoR-PhoP involving modification of pathway-specific regulators.     

A gene cloning system for the aranciamycin producer strain Streptomyces echinatus Lv 22

Streptomyces echinatus Lv 22 (=DSM 40730) produces an anthracycline antibiotic aranciamycin. Development of DNA transfer using conjugation from Escherichia coli into this strain is described. Various replicative plasmids (pKC1139, pKC1218E, pSOK1O1, pCHZ101) as well as actinophage (φC31- and VWB-based vectors pSET152 and pVWB, respectively, were transferred from E. coli ET12567 (pUB307) into the S. echinatus at a frequency ranging from 2.4 × 10^?3 to 1.6 × 10^?4. The transconjugants did not differ from wild type in their ability to produce aranciamycin and morphological features. There is one attB site for pSET152 and pVWB integrative plasmids in the S. echinatus chromosome. Developed DNA transfer system was used for expression of heterologous regulatory genes in S. echinatus cells. Expression of relA gene of ppGpp synthetase increased antibiotic production in S. echinatus. The absA2 gene of S. ghanaensis appears to play a negative role in the control of aranciamycin biosynthesis. Additional copies of absA2 leads to inhibition of aranciamycin’s production. absB and afsS had no effect on aranciamycin biosynthesis as well as on the morphological features of S. echinatus. Obtained results indicate efficiency of the developed system for gene cloning in S. echinatus.     

An efficient approach for cloning the dNDP-glucose synthase gene from actinomycetes and its application in Streptomyces spectabilis, a spectinomycin producer

Specifically designed PCR primers were applied to amplify a segment of dTDP-glucose synthase gene from six actinomycete strains. About 300-bp or 580-bp DNA fragments were obtained from all the organisms tested. By DNA sequence analysis, seven amplified fragments showed high homology with dTDP-glucose synthase genes that participate in the biosynthesis of secondary metabolites or in deoxy-sugar moieties in lipopolysaccharides. In addition, we have cloned a 45-kb region of DNA from Streptomyces spectabilis ATCC27741, a spectinomycin producer which contained the dTDP-glucose synthase and dTDP-glucose 4,6-dehydratase genes named spcD and spcE, respectively. The spcE gene was expressed in Escherichia coli and the activity was assayed in cell extracts. The enzyme showed substrate specificity only to dTDP-glucose.     

Insertional inactivation of mtrX and mtrY genes from the mithramycin gene cluster affects production and growth of the producer organism Streptomyces argillaceus

Mithramycin is an antitumor aromatic polyketide synthesized by Streptomyces argillaceus. Two genes (mtrX and mtrY) of the mithramycin gene cluster were inactivated by gene replacement. Inactivation of mtrX, that encodes an ABC excision nuclease system for DNA repair, produced a mutant that was affected in the normal rate of growth. Expression of mtrX in Streptomyces albus in a multicopy plasmid vector conferred a low increase in resistance to mithramycin. Inactivation of mtrY, that encodes a protein of unknown function, produced a 50% decrease in mithramycin biosynthesis. When mtrY was expressed in the wild-type S. argillaceus in a multicopy plasmid, this caused about 47% increase in the levels of mithramycin production. It is proposed that mtrX and mtrY could code for a secondary defense mechanism and a mithramycin regulatory element, respectively.     

An inducible Streptomyces gene cluster involved in aromatic compound metabolism

Streptomyces setonii (ATCC 39116) is a thermophilic soil actinomycete capable of degrading single aromatic compounds including phenol and benzoate via the ortho-cleavage pathway. Previously, a 6.3-kb S. setonii DNA fragment containing a thermophilic catechol 1,2-dioxygenase (C12O) gene was isolated and functionally overexpressed in Escherichia coli (An et al., FEMS Microbiol. Lett. 195 (2001) 17-22). Here the 6.3-kb S. setonii DNA fragment was shown to be organized into two putative divergently transcribed gene clusters with six complete and one incomplete open reading frames (ORFs). The first cluster with three ORFs showed homologies to previously known benA, benB, and benC, implying it is a part of the benzoate catabolic operon. The second cluster revealed an ortho-cleavage catechol catabolic operon with three translationally coupled ORFs (in order): catR, a putative LysR-type regulatory gene; catB, a muconate cycloisomerase gene; catA, a C12O gene. Each of these individually cloned ORFs was expressed in E. coli and identified as a distinct protein. The expression of the cloned S. setonii catechol operon was induced in Streptomyces lividans by specific single aromatic compounds including catechol, phenol, and 4-chlorophenol. A similar induction pattern was also observed using a luciferase gene-fused reporter system.     

An efficient method for the overexpression and purification of active tyrosinase from Streptomyces castaneoglobisporus

The melanin-synthesizing gene operon cloned from Streptomyces castaneoglobisporus HUT6202 consists of two genes, designated tyrC and orf378, which encode apotyrosinase (TYRC) and its activator protein (ORF378), respectively. We have suggested that ORF378 may facilitate the incorporation of Cu(II) into apotyrosinase to express tyrosinase activity. To overproduce ORF378 and TYRC in Escherichia coli BL21(DE3)-pLysS, tyrC, and orf378 were independently but not polycistronically placed under the control of a T7 promoter in a vector, pET-21a(+). His(6)-tagged TYRC and His(6)-tagged ORF378 were simultaneously overproduced in an E. coli strain harboring a plasmid, designated pET-mel2, and the two proteins were co-purified with a Ni(II)-bound affinity column. Gel filtration analysis revealed that the two proteins form a heterodimer complex. The complexed protein was retrieved at a high efficiency (11 mg/L). To obtain an active TYRC, which is a Cu(II)-bound form of tyrosinase, we constructed pET-mel3 that carries orf378 without His(6)-tag and His(6)-tagged tyrC. After the cell-free extract from E. coli harboring pET-mel3 was subjected to Cu(II)-bound affinity column chromatography, His(6)-tagged TYRC, eluted from the column, exhibited the tyrosinase activity. The k(cat) and K(m) values for l-3,4-dihydroxyphenylalanine (l-DOPA) of His(6)-tagged TYRC, which catalyzes the oxidation of l-DOPA to dopaquinone, were 880+/-80s(-1) and 8.1+/-0.9 mM, respectively.     

无机盐对阿维链霉菌接合转移及异源表达放线紫红素的影响

摘要：【目的】阿维链霉菌可作为异源表达抗生素生物合成基因簇的良好宿主,但是需要优化含有大片段DNA质粒的接合转移效率。【方法】我们选取MgCl2、NaCl、Ca(NO3)2 和CaCl2等4种无机盐,在0－200 mmol/L浓度范围内分别研究其对大质粒向阿维链霉菌接合转移的影响,再设计完全随机试验筛选最佳条件。【结果】CaCl2对阿维链霉菌接合转移有极明显的促进作用,MgCl2也有一定提高作用。通过完全随机试验筛选出最佳的CaCl2和MgCl2浓度组合,使大质粒的接合转移效率提高11倍。同时,本研究还发现阿维链霉菌异源表达放线紫红素的最适培养基,成功表达放线紫红素。【结论】特定无机盐对阿维链霉菌接合转移效率有明显提高作用,并且能促进放线紫红素在阿维链霉菌中的表达。     

An efficient gene transfer system for hematopoietic cell line using transient and stable vectors

The hematopoietic system represents an interesting model for gene transfer protocols. Here, we have evaluated the efficiency of a gene transfer system using the polycationic compound SuperFect (Qiagen) and the K562 hematopoietic cell line. Transient and stable vectors carrying the enhanced green fluorescent protein (EGFP) reporter gene were employed. The stable vector was constructed based on Epstein-Barr virus sequences such as EBV oriP (origin of replication) and EBNA (EBV nuclear antigen)-1, both for DNA replication. The transfection efficiency of the viable cells was estimated by flow cytometry at approximately 98% for transient and stable vectors. Transiently transfected cells presented optimal EGFP expression until day 2 when fluorescence started to decrease. In contrast, stable transfectants continuously expressed the marker gene product for 10 weeks in the presence of G418. Our results represent an efficient gene transfer method for K562 hematopoietic cells and may be used as an alternative approach for further gene transfer studies involving hematopoietic cells.     

A two-vector system for the production of recombinant polyketides in Streptomyces

A two-vector system was developed for heterologous expression of the three genes comprising the 6-deoxyerythronolide B synthase (DEBS) polyketide gene cluster. Individual DEBS genes and pairwise combinations of two such genes were each cloned downstream of the actinorhodin (actI) promoter in two compatible Streptomyces vectors: the autonomously replicating vector, pKAO127′Kan′, and the integrating vector, pSET152. The resulting plasmids were either simultaneously or sequentially transformed into Streptomyces lividans K4-114. Efficient trans-complementation of modular polyketide synthase subunit proteins occurred when the respective genes were transcribed from the two vectors and resulted in production of the erythromycin precursor 6-deoxyerythronolide B (6-dEB). Journal of Industrial Microbiology & Biotechnology (2000) 24, 46–50. Received 17 March 1999/ Accepted in revised form 15 September 1999     

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.     

Nanoparticulate system for efficient gene transfer into refractory cell targets

A biocompatible, nanoparticulate formulation has been designed to retain, protect, and deliver adenoviral gene constructs over an extended time course. Such devices can be administered locally or systemically with low toxicity. A multipolymeric nanoparticulate system, featuring very high stability in physiologic media, was designed to allow efficient in vitro gene transfer. The efficacy of nanoparticulate delivery is effective in cell systems that are normally refractory to gene transfer, such as pancreatic islets and antigen-presenting cells. The findings suggest a nonspecific uptake system that permits adenoviral particle release within the transfected cells. A comparison with literature data revealed that our system is efficient at much lower levels (at least three orders of magnitude) of infectious viral particles.