Development of an Intergeneric Conjugal Transfer System for Xinaomycins-Producing Streptomyces noursei Xinao-4

Sun, Feng-Hui; Luo, Dejun; Shu, Dan; Zhong, Juan; Tan, Hui

doi:10.3390/ijms150712217

Cited by 10 publications

(10 citation statements)

References 40 publications

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“…Restriction-modification system is an obstacle of introducing the plasmid into Gram-positive strains. The heat shock is usually applied to reduce the activity of a host restriction-modification system and to improve transformation efficiency [ 24 , 36 , 37 , 38 ]. To determine whether the heat shock would improve the electroporation efficiency, a water bath suitable for Gram-positive strains was performed after electroporation according to previous reports [ 11 , 24 ].…”

Section: Resultsmentioning

confidence: 99%

Development of an Efficient Electroporation Method for Iturin A-Producing Bacillus subtilis ZK

Zhang

Ding

Shu

et al. 2015

IJMS

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In order to efficiently introduce DNA into B. subtilis ZK, which produces iturin A at a high level, we optimized seven electroporation conditions and explored an efficient electroporation method. Using the optimal conditions, the electroporation efficiency was improved to 1.03 × 107 transformants/μg of DNA, an approximately 10,000-fold increase in electroporation efficiency. This efficiency is the highest electroporation efficiency for B. subtilis and enables the construction of a directed evolution library or the knockout of a gene in B. subtilis ZK for molecular genetics studies. In the optimization process, the combined effects of three types of wall-weakening agents were evaluated using a response surface methodology (RSM) design, which led to a two orders of magnitude increase in electroporation efficiency. To the best of our limited knowledge, this study provides the first demonstration of using an RSM design for optimization of the electroporation conditions for B. subtilis. To validate the electroporation efficiency, a case study was performed and a gene (rapC) was inactivated in B. subtilis ZK using a suicide plasmid pMUTIN4. Moreover, we found that the rapC mutants exhibited a marked decrease in iturin A production, suggesting that the rapC gene was closely related to the iturin A production.

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Section: Resultsmentioning

confidence: 99%

Development of an Efficient Electroporation Method for Iturin A-Producing Bacillus subtilis ZK

Zhang

Ding

Shu

et al. 2015

IJMS

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“…Theoretically, the described procedure provides a promising approach for the ultimate site-specific homologous recombination between this cassette and the bacterial chromosome to generate targeted gene knockouts. However, in practice, it does not work properly for every Streptomyces species, and further optimization is often required to successfully conjugate E. coli with refractory species (62)(63)(64)(65). Various species-dependent parameters which strongly affected the transformation efficiency were identified.…”

Section: Discussionmentioning

confidence: 99%

“…Various species-dependent parameters which strongly affected the transformation efficiency were identified. Sun et al (64) found that the ratio of donor to recipient cell numbers was the most influential factor for conjugation into Streptomyces noursei. Also, for Streptomyces diastatochromogenes, the number of spores and their use instead of mycelia, in combination with a cultivation step at 30°C subsequent to the heat shock, were found to be the most important factors (65).…”

Section: Discussionmentioning

confidence: 99%

“…In that study, tryptic soy agar medium containing 10 mmol/liter MgCl 2 was the preferred medium for conjugation. Finally, Sun et al (64) obtained 8 ϫ 10 Ϫ3 exconjugants per recipient when spores were heat shocked at 50°C for 10 min, mixed with E. coli ET12567/pUZ8002/ pSET152 in the ratio of 1:100, plated on 2CMY medium (71) containing 40 mmol/liter MgCl 2 , and incubated at 30°C for 22 h.…”

Section: Discussionmentioning

confidence: 99%

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An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35

Netzker

Schroeckh

Gregory

et al. 2016

Appl Environ Microbiol

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Streptomyces iranensis HM 35 is an alternative rapamycin producer to Streptomyces rapamycinicus. Targeted genetic modification of rapamycin-producing actinomycetes is a powerful tool for the directed production of rapamycin derivatives, and it has also revealed some key features of the molecular biology of rapamycin formation in S. rapamycinicus. The approach depends upon efficient conjugational plasmid transfer from Escherichia coli to Streptomyces, and the failure of this step has frustrated its application to Streptomyces iranensis HM 35. Here, by systematically optimizing the process of conjugational plasmid transfer, including screening of various media, and by defining optimal temperatures and concentrations of antibiotics and Ca 2؉ ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions in S. iranensis HM 35. Among them were rapK, which generates the starter unit for rapamycin biosynthesis, and hutF, encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants of Streptomyces species that are genetically difficult to manipulate. IMPORTANCE The developed protocol of conjugational plasmid transfer from Escherichia coli to Streptomyces iranensis may allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable, Streptomyces species. Since the discovery of streptomycin in 1943 (1), streptomycetes have been shown to produce thousands of compounds with possibly beneficial features, e.g., antibiotics, immunosuppressants, or anticancer drugs. Actinomycete-derived metabolites comprise over two-thirds of all known antibiotic compounds (2), and recent genome sequencing programs revealed that their biosynthesis potential has been underestimated. In their 8-to 12-Mb genomes, approximately 20 to 30 gene clusters encode the biosynthesis of secondary metabolites (3-5). One of the most important Streptomyces-derived compounds is the mechanistic target of rapamycin (mTOR) inhibitor rapamycin (sirolimus), a "billion dollar molecule" and, after cyclosporine, the most widely used immunosuppressant of microbial origin (6). Rapamycin was previously reported as an antifungal antibiotic produced by Streptomyces hygroscopicus ATCC 29253 (7), later renamed Streptomyces rapamycinicus (8). The rapamycin gene cluster in this strain has been sequenced (9), the biosynthetic pathway has been extensively characterized (10-14), and engineering of the cluster has yielded an impressive range of bioactive modified rapamycins (rapalogs) (15, 16), some in multigram amounts. So far, two other rapamycin-producing species are known: the taxonomically closely related Streptomyces iranensis HM 35 (5, 17) and Actinoplanes sp. strain N902-109 (18). To elucidate the molecular biology of rapamycin formation in these strains and to further exploit the physiological and pharmacological capability of rapamycin derivatives, genetic manipulation of these altern...

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“…The selection of an appropriate conjugation medium has an obvious influence on the conjugation efficiency in streptomycetes (Du et al, 2012;Sun et al, 2014), so three applicable solid media, MS, TSB, and 2CMC, were tested to determine the appropriate medium. The 2CMC medium proved to be optimal for the intergeneric conjugation of E. coli-S. rimosus M527, whether mycelia or spores were chosen as recipient (Table 2).…”

Section: Development and Optimization Of Intergeneric Conjugation Tramentioning

confidence: 99%

Development and optimization of an intergeneric conjugation system and analysis of promoter activity in Streptomyces rimosus M527

Song

Liao

et al. 2019

J. Zhejiang Univ. Sci. B

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An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes. In this study, firstly, a genetic transformation system based on intergeneric conjugation was developed in Streptomyces rimosus M527, a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi. Some experimental parameters involved in this procedure were optimized, including the conjugative media, ratio of donor to recipient, heat shock temperature, and incubation time of mixed culture. Under the optimal conditions, a maximal conjugation frequency of 3.05×10 −5 per recipient was obtained. Subsequently, based on the above developed and optimized transformation system, the synthetic promoters SPL-21 and SPL-57, a native promoter potrB, and a constitutive promoter permE * commonly used for gene expression in streptomycetes were selected and their activity was analyzed using gusA as a reporter gene in S. rimosus M527. Among the four tested promoters, SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in β-glucuronidase (GUS) activity compared with the control promoter permE *. Promoter SPL-57 showed activity comparable to that of permE *. Promoter potrB, which showed the lowest activity, showed a 50% decrease in GUS activity compared with the control permE *. The transformation system developed in this study and the tested promotors provide a basis for the further modification of S. rimosus M527.

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Development of an Intergeneric Conjugal Transfer System for Xinaomycins-Producing Streptomyces noursei Xinao-4

Cited by 10 publications

References 40 publications

Development of an Efficient Electroporation Method for Iturin A-Producing Bacillus subtilis ZK

Development of an Efficient Electroporation Method for Iturin A-Producing Bacillus subtilis ZK

An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35

Development and optimization of an intergeneric conjugation system and analysis of promoter activity in Streptomyces rimosus M527

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