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。     

Interspecies conjugation of Escherichia coli-Streptomyces globisporus 1912 using integrative plasmid pSET152 and its derivatives]

Streptomyces globisporus 1912 produces a novel angucycline antitumor antibiotic landomycin E (LE). To study the LE biosynthetic gene cluster in detail, a system for the conjugal transfer of the integrative plasmid pSET152 from Escherichia coli into S. globisporus 1912 has been developed. It was shown that this plasmid integrates into two sites of the S. globisporus chromosome and is stably inherited under nonselective conditions. pSET152+ exconjugants of the strain 1912 are characterized by a significant decrease in LE synthesis (by 50-90%). A negative effect of pSET152 integration on antibiotic production was observed even upon the use of the recipient strain with increased LE synthesis, although in this case, the level of LE production in ex-conjugants was 120-150% of that in the original strain 1912. Based on pSET152, a vector system for gene knockouts in S. globisporus was developed. The effectivity of this system was shown in the example of disruption of the lndA gene encoding the key enzyme of LE synthesis (beta-ketoacylsynthase). Inactivation of this gene was shown to lead to the cessation of LE biosynthesis.     

浅蓝霉素产生菌海洋异壁放线菌WH1-2216-6 遗传操作体系的建立

摘要: 【目的】建立二联吡啶类抗生素浅蓝霉素的产生菌海洋异壁放线菌Actinoalloteichus sp. WH1-2216-6的遗传操作体系,以便对浅蓝霉素的相关生物合成基因进行体内敲除和基因回补等遗传操作。【方法】以整合型质粒pSET152 为外源DNA,探索和优化了异壁放线菌WH1-2216-6 菌株与大肠杆菌进行接合转移实验的方法和条件,以此为基础,利用PCR-targeting 系统在体外构建了一个浅蓝霉素二羟基苯甲酸甲酯AMP 连接酶基因敲除的cosmid 质粒pCSG2104,并在优化后的     

Intergeneric conjugation in Streptomyces peucetius and Streptomyces sp. strain C5: chromosomal integration and expression of recombinant plasmids carrying the chiC gene

Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces circumvents problems such as host-controlled restriction and instability of foreign DNA during the transformation of Streptomyces protoplasts. The anthracycline antibiotic-producing strains Streptomyces peucetius and Streptomyces sp. strain C5 were transformed using E. coli ET12567(pUZ8002) as a conjugal donor. When this donor species, carrying pSET152, was mated with Streptomyces strains, the resident plasmid was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. Analysis of the exconjugants showed stable integration of the plasmid at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the chromosomal integration site was determined and shown to be conserved. However, the core sequence, where the crossover presumably occurred in C5 and S. peucetius, is TTC. These results also showed that the phiC31 integrative recombination is active and the phage attP site is functional in S. peucetius as well as in C5. The efficiency and specificity of phiC31-mediated site-specific integration of the plasmid in the presence of a 3.7-kb homologous DNA sequence indicates that integrative recombination is preferred under these conditions. The integration of plasmid DNA did not affect antibiotic biosynthesis or biosynthesis of essential amino acids. Integration of a single copy of a mutant chiC into the wild-type S. peucetius chromosome led to the production of 30-fold more chitinase.     

Efficient DNA transformation of Bradyrhizobium japonicum by electroporation.

Intact cells of Bradyrhizobium japonicum USDA 110 were transformed with a 30-kilobase plasmid to efficiencies of 10(6) to 10(7) transformants per microgram by high-voltage electroporation. The technique was reliable and simple, with single colonies arising from transformed cells within 5 days of antibiotic selection. Plasmid DNA from B. japonicum transformed the Bradyrhizobium (Arachis) sp. with high efficiency, while the same plasmid extracted from Escherichia coli transformed B. japonicum at very low efficiency. The electrical conditions that resulted in the highest efficiencies were high voltage (10.5 to 12.5 kV/cm) and short pulse length (6 to 7 ms). A linear increase in the number of transformants was observed as DNA concentration was increased over 4 orders of magnitude; saturation appeared to begin between 120 ng/ml and 1.2 micrograms/ml. This novel method of transformation should enhance B. japonicum genetic research by providing a valuable alternative to conjugal mating, which is currently the only efficient, widely used means of introducing DNA into this organism.     

Efficient DNA transformation of Bradyrhizobium japonicum by electroporation

Intact cells of Bradyrhizobium japonicum USDA 110 were transformed with a 30-kilobase plasmid to efficiencies of 10(6) to 10(7) transformants per microgram by high-voltage electroporation. The technique was reliable and simple, with single colonies arising from transformed cells within 5 days of antibiotic selection. Plasmid DNA from B. japonicum transformed the Bradyrhizobium (Arachis) sp. with high efficiency, while the same plasmid extracted from Escherichia coli transformed B. japonicum at very low efficiency. The electrical conditions that resulted in the highest efficiencies were high voltage (10.5 to 12.5 kV/cm) and short pulse length (6 to 7 ms). A linear increase in the number of transformants was observed as DNA concentration was increased over 4 orders of magnitude; saturation appeared to begin between 120 ng/ml and 1.2 micrograms/ml. This novel method of transformation should enhance B. japonicum genetic research by providing a valuable alternative to conjugal mating, which is currently the only efficient, widely used means of introducing DNA into this organism.     

Improvement of transformation and electroduction in avermectin high-producer,<Emphasis Type="Italic">Streptomyces avermitilis</Emphasis>

Factors affecting the PEG-mediated transformation and electrotransformation of Streptomyces avermitilis protoplasts, an industrial avermectin high-producer, were evaluated. The maximum protoplast transformation efficiency under optimum conditions with PEG was 3 x 106 transformants per microg plasmid pIJ702 DNA. The efficiency of electrotransformation with the same plasmid the intact cells grown in medium with 0.5 mmol/L CaCl2, suspended in buffer with 0.5 mol/L sucrose +1 mmol/L MgCl2, and pulsed at an electric field strength of 10 kV/cm, 800 ohms, 25 microF, was of 2 x 10(3) transformants per microg DNA. When the cells were electroporated after mild lysozyme-treatment, the efficiency was up to 10(4) transformants per microg DNA. Electroporation of protoplasts and germlings had a lower efficiency (10(2) transformants per microg DNA). We report that electroporation under optimum conditions can be used for direct transfer of nonconjugative plasmid pIJ699 between two different Streptomyces species, S. avermitilis and S. lividans.     

High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes

Many streptomycetes, including S. coelicolor A3(2), possess a potent methyl-specific restriction system which can present an effective barrier to the introduction of heterologous DNA. We have compared the efficiency of intergeneric conjugal transfer of different types of plasmids to S. coelicolor and S. lividans 66 using two E. coli donors: the standard, methylation proficient strain S17-1. and the methylation deficient donor, ET12567(pUB307). We demonstrate that the methylation deficient donor can yield > 10⁴-fold more S. coelicolor exconjugants than the standard donor. In the case of pSET152 derivatives, which integrate into the host chromosome by site-specific recombination, up to 10% of streptomycete spores in the conjugation mixture inherit the plasmid. The conjugation procedure is efficient enough to obtain exconjugants with 'suicide' delivery plasmids and therefore provides a simple route for conducting gene disruptions in methyl DNA-restricting streptomycetes, and possibly other bacteria.     

Methods for the genetic manipulation of <Emphasis Type="Italic">Nonomuraea</Emphasis> sp. ATCC 39727

Nonomuraea sp. ATCC 39727 belongs to the Streptosporangiaceae family of filamentous actinomycetes. This microorganism produces the teicoplanin-like glycopeptide A40926, which is the starting material for the synthesis of the second-generation glycopeptide dalbavancin. Notwithstanding the strain’s pharmaceutical relevance, the lack or poor efficiency of genetic tools to manipulate Nonomuraea sp. ATCC 39727 has hampered strain and product improvement. Here we report the development of gene transfer systems based on protoplast transformation and intergeneric conjugation from Escherichia coli. Efficiency of transformation and conjugation, followed by site specific or homologous recombination with the Nonomuraea sp. genome, were determined using the integrative plasmid pSET152 (5.7 kb), and the Supercos1 derivative cosmid A40ΔY (30 kb). To our knowledge, this is the first report of the transformation of protoplasts of Nonomuraea sp. ATCC 39727, even though the improved procedure for intergeneric conjugation makes it the method of choice for introducing large segments of DNA into Nonomuraea sp. ATCC 39727.     

Liposome-enhanced transformation of streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of streptococcus lactis and bacillus subtilis

Abstract An efficient protoplast transformation system and a procedure of plasmid transfer by means of protoplast fusion is described for Streptococcus lactis . Protoplasts of S. lactis IL1403 and S. lactis MG1363 were transformed by pGK12 [2.9 MDa erythromycin resistance (Em^r)] with an efficiency of 3 × 10⁵ transformants per μg plasmid DNA. This high efficiency was obtained by the inclusion in the transformation mixture of liposomes composed of cardiolipin and phosphatidyl choline in a molar ratio of 1 to 6 in the presence of 22.5% polyethylene glycol (PEG). This paper also reports an efficient plasmid transfer method between lactic and streptococci and Bacillus subtilis by means of protoplast fusion. When S. lactis and B. lactis protoplasts undergo fusion mediated by exposure to 37.5% polyethylene glycol, plasmid pGKV21 (3.2 MDa; Em^r) was transfered from one host to the other with a frequency of 10⁻³−10⁻⁵ transformants per regenerating recipient protoplast.     

Intergeneric Conjugation Escherichia coli–Streptomyces globisporus1912 Using Integrative Plasmid pSET152 and Its Derivatives

Streptomyces globisporus1912 produces a novel angucycline antitumor antibiotic landomycin E (LE). To study the LE biosynthetic gene cluster in detail, a system for the conjugal transfer of the integrative plasmid pSET152 fromEscherichia coliinto S. globisporus1912 has been developed. It was shown that this plasmid integrates into two sites of the S. globisporuschromosome and is stably inherited under nonselective conditions. pSET152⁺exconjugants of the strain 1912 are characterized by a significant decrease in LE synthesis (by 50–90%). A negative effect of pSET152 integration on antibiotic production was observed even upon the use of the recipient strain with increased LE synthesis, although in this case, the level of LE production in exconjugants was 120–150% of that in the original strain 1912. Based on pSET152, a vector system for gene knockouts in S. globisporuswas developed. The effectivity of this system was shown in the example of disruption of the lndAgene encoding the key enzyme of LE synthesis (-ketoacylsynthase). Inactivation of this gene was shown to lead to the cessation of LE biosynthesis.     

Partial characterization of the genetic basis for sucrose metabolism and nisin production in Streptococcus lactis.

We attempted to identify the genetic loci for sucrose-fermenting ability (Suc+), nisin-producing ability (Nip+), and nisin resistance (Nisr) in certain strains of Streptococcus lactis. To obtain genetic evidence linking the Suc+ Nip+ Nisr phenotype to a distinct plasmid, both conjugal transfer and transformation were attempted. A conjugation procedure modified to protect the recipients against the inhibitory action of nisin allowed the conjugal transfer of the Suc+ Nip+ Nisr marker from three Suc+ Nip+ Nisr donors to various recipients. The frequency of transfer ranged from 1.7 x 10(-4) to 5.6 x 10(-8) per input donor, depending on the mating pair. However, no additional plasmid DNA was apparent in these transconjugants. Transformation of S. lactis LM0230 to the Suc+ Nip+ Nisr phenotype by using the plasmid pool of S. lactis ATCC 11454 was not achieved, even though other plasmids present in the pool were successfully transferred. However, two results imply the involvement of plasmid DNA in coding for the Suc+ Nip+ Nisr phenotype. The Suc+ Nip+ Nisr marker was capable of conjugal transfer to a recipient deficient in host-mediated homologous recombination (Rec-), and the Suc+ Nip+ Nisr marker exhibited bilateral plasmid incompatibility with a number of lactose plasmids found in S. lactis. Although our results indicate that the Suc+ Nip+ Nisr phenotype is plasmid encoded, no physical evidence linking this phenotype to a distinct plasmid was obtained.     

委内瑞拉链霉菌秦岭变种属间接合转移系统的构建及透明颤菌血红蛋白的表达

【目的】构建委内瑞拉链霉菌秦岭变种属间接合转移系统及透明颤菌血红蛋白的表达。【方法】以链霉菌广泛使用的整合型质粒pSET152和复制型pHZ1358为出发质粒,通过供体大肠杆菌(Escherichia coli)ET12567(pUZ8002)进行属间接合转移委内瑞拉链霉菌秦岭变种。【结果】确定了该变种的最佳接合转移条件;通过SOE-PCR(Splicing by overlap extension PCR)技术构建含PermE和vhb结构基因融合片段的整合型表达载体pJD100,转化ET12567(pUZ8002)后属间接合转移委内瑞拉链霉菌秦岭变种。通过PCR和CO结合差光谱验证了vhb基因在委内瑞拉链霉菌秦岭变种中的整合表达。【结论】本文首次探索了委内瑞拉链霉菌秦岭变种接合转移系统,确定了委内瑞拉链霉菌秦岭变种的最佳接合转移条件,并采用基因工程手段使vhb基因在委内瑞拉链霉菌秦岭变种中获得表达。     

Partial characterization of the genetic basis for sucrose metabolism and nisin production in Streptococcus lactis

We attempted to identify the genetic loci for sucrose-fermenting ability (Suc+), nisin-producing ability (Nip+), and nisin resistance (Nisr) in certain strains of Streptococcus lactis. To obtain genetic evidence linking the Suc+ Nip+ Nisr phenotype to a distinct plasmid, both conjugal transfer and transformation were attempted. A conjugation procedure modified to protect the recipients against the inhibitory action of nisin allowed the conjugal transfer of the Suc+ Nip+ Nisr marker from three Suc+ Nip+ Nisr donors to various recipients. The frequency of transfer ranged from 1.7 x 10(-4) to 5.6 x 10(-8) per input donor, depending on the mating pair. However, no additional plasmid DNA was apparent in these transconjugants. Transformation of S. lactis LM0230 to the Suc+ Nip+ Nisr phenotype by using the plasmid pool of S. lactis ATCC 11454 was not achieved, even though other plasmids present in the pool were successfully transferred. However, two results imply the involvement of plasmid DNA in coding for the Suc+ Nip+ Nisr phenotype. The Suc+ Nip+ Nisr marker was capable of conjugal transfer to a recipient deficient in host-mediated homologous recombination (Rec-), and the Suc+ Nip+ Nisr marker exhibited bilateral plasmid incompatibility with a number of lactose plasmids found in S. lactis. Although our results indicate that the Suc+ Nip+ Nisr phenotype is plasmid encoded, no physical evidence linking this phenotype to a distinct plasmid was obtained.     

Effect of various irradiation treatments of plant protoplasts on the transformation rates after direct gene transfer

Summary In P. hybrida and B. nigra an enhancement of transformation rates (direct gene transfer) of about six to seven-fold was obtained after irradiation of protoplasts with 12.5 Gy (X-ray). The effect of protoplast irradiation was similar in experiments where protoplasts were irradiated 1h before transformation (X-ray/DNA) or 1h after completion of the transformation procedure (DNA/X-ray). Increased X-ray doses up to 62.5 Gy resulted in further enhancement of percentages of transformed colonies, indicating a correlation between relative transformation frequencies (RTF) and the doses applied. Estimation of degradation rates of plasmid sequences in plant protoplasts yielded a reduction of plasmid concentration to 50% 8–12 h after transformation. In 1-day-old protoplasts, the level of plasmid fragments dropped to 0%–10% compared to 1h after transformation. The results demonstrate that the integration rates of plasmid sequences into the plant genome may in part be governed by DNA repair mechanisms. This could be an explanation for the observed genotypic dependence of transformation rates in different plant species and plant genotypes. Gene copy number reconstructions revealed enhanced integration rates of plasmid sequences in transformed colonies derived from irradiated protoplasts.     

Promotion of spinosad biosynthesis by chromosomal integration of the Vitreoscilla hemoglobin gene in Saccharopolyspora spinosa

To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the control of the promoter for the erythromycin resistance gene by splicing using overlapping extension PCR. This was cloned into the integrating vector pSET152, yielding the Vitreoscilla hemoglobin gene expression plasmid pSET152EVHB. This was then introduced into S. spinosa SP06081 by conjugal transfer, and integrated into the chromosome by site-specific recombination at the integration site ΦC31 on pSET152EVHB. The resultant conjugant, S. spinosa S078-1101, was genetically stable. The integration was further confirmed by PCR and Southern blotting analysis. A carbon monoxide differential spectrum assay showed that active Vitreoscilla hemoglobin was successfully expressed in S. spinosa S078-1101. Fermentation results revealed that expression of the Vitreoscilla hemoglobin gene significantly promoted spinosad biosynthesis under normal oxygen and moderately oxygen-limiting conditions (P<0.01). These findings demonstrate that integrating expression of the Vitreoscilla hemoglobin gene improves oxygen uptake and is an effective means for the genetic improvement of S. spinosa fermentation.     

黑暗链霉菌DNA转移系统的建立

研究了黑暗链霉菌的基因转移系统,探索了通过PEG介导的原生质体转化、接合转移向黑暗链霉菌中转入外源DNA的可能性。多次尝试用质粒pIJ702转化黑暗链霉菌9904原生质体均未成功。对原生质体进行“热处理”后转化、利用单链DNA转化等都不能将质粒导入黑暗链霉菌中,表明黑暗链霉菌对外源DNA有很强的限制修饰作用。利用接合转移将具有oriT的大肠杆菌链霉菌穿梭质粒pHZ132转入大肠杆菌ET12567(pUZ8002)中,获得供体菌ET12567(pUZ8002,pHZ132)。将供体菌与预萌发的黑暗链霉菌9904的孢子进行接合转移,成功地将pHZ132转入黑暗链霉菌9904中。质粒pHZ132经黑暗链霉菌自身修饰后也可转入黑暗链霉菌9904菌株的原生质体中,转化率约为103/μg DNA(pHZ132)。     

藤黄生孢链霉菌NRRL 2401遗传操作系统和基因文库的构建北大核心CSCD

【目的】建立藤黄生孢链霉菌NRRL 2401的遗传操作系统和基因文库,以便筛选次级代谢产物生物合成基因。【方法】利用大肠杆菌和链霉菌的属间接合转移的方法,以整合型载体pPM927、pSET152和复制型载体pJTU1278构建链霉菌遗传操作系统。以pCClFOS^(TM)载体,大肠杆菌EP1300^(TM)-T1~R为宿主菌构建Fosmid文库。随后,设计引物,利用"板池-行池-单克隆"的三级PCR方法对文库进行快速筛选。【结果】pPM927、pSET152和pJTU1278均成功转入藤黄生孢链霉菌NRRL 2401,其中pSET152载体的转化效率最高。构建了稳定高效的藤黄生孢链霉菌NRRL 2401的基因文库,含2880个克隆,平均插入片段大小约为35 kb,空载率小于1%,文库覆盖率为99.99%,覆盖基因组16.5倍。同时,初步筛选出可能含有吲哚霉素生物合成基因簇的9个阳性克隆。【结论】成功构建了稳定高效的藤黄生孢链霉菌NRRL 2401遗传操作系统和高质量的基因文库,为克隆该菌中次级代谢产物生物合成基因簇以及进一步遗传改造奠定了基础。     

Protoplast transformation of recalcitrant alkaliphilic Bacillus sp. with methylated plasmid DNA and a developed hard agar regeneration medium

Among the diverse alkaliphilic Bacillus strains, only a little have been reported to be genetically transformed. In this study, an efficient protoplast transformation procedure was developed for recalcitrant alkaliphilic Bacillus sp. N16-5. The procedure involved polyethylene glycol-induced DNA uptake by the protoplasts and subsequent protoplast regeneration with a developed hard agar regeneration medium. An in vivo methylation strategy was introduced to methylate the exogenous plasmid DNA for improving the transformation efficiency. The transformation efficiency reached to 1.1×10(5) transformants per μg plasmid DNA with methylated plasmid pHCMC04 and the developed hard agar regeneration medium. This procedure might also be applicable to the genetic transformation of other Bacillus strains.     

Transformation of the Pseudonocardiaceae Amycolatopsis sp. strain HR167 is highly dependent on the physiological state of the cells

The Pseudonocardiaceae Amycolatopsis sp. strain HR167 is used in a biotransformation process to produce vanillin from ferulic acid. To make this strain accessible for genetic engineering, a direct mycelium transformation system developed for Amycolatopsis mediterranei [Madon and Hotter (1991) J Bacteriol 173: 6325-6331] was applied and optimized for Amycolatopsis sp. strain HR167. The physiological state of the cells had a major influence on the transformation rate. The highest transformation rate of about 7x10(5) transformants per microgram of DNA was obtained with mycelium harvested 6.5-7.5 h after the culture has reached the stationary growth phase. When cells were harvested outside of this time slot, the transformation rate drastically decreased. The density of the mycelium suspensions used in the transformation mixture and the methylation state of the plasmid DNA used for the transformation were also crucial parameters. With plasmid DNA isolated from Escherichia coli ET12567, transformation rates were 3,500-fold higher than those obtained with DNA isolated from E. coli XL1-Blue.