Development of an efficient conjugal DNA transfer system between Escherichia coli and a non-sporulating Streptomyces strain

Rocha, Diana; Ruíz-Villafán, Beatriz; Manzo-Ruiz, Monserrat; Rodríguez‐Sanoja, Romina; Sánchez, Sergio

doi:10.1016/j.mimet.2017.11.006

Cited by 7 publications

(4 citation statements)

References 34 publications

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“…Recently, mycelia have shown potency in intergeneric conjugation. Some research results concerning intergeneric conjugation using mycelia as recipient have been reported for several Streptomyces (Du et al, 2012;Rocha et al, 2018;Zhang et al, 2018). The conjugation conditions were highly strain-specific and usually ineffective for different actinobacteria.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…Transformation systems for several Streptomyces strains have been developed. Rocha (Rocha, Ruiz‐Villafán, & Manzo, ) reported a reliable genetic system for Streptomyces peucetius var. caesius , and the optimal conjugation frequency was up to 5 × 10 −4 .…”

Section: Discussionmentioning

confidence: 99%

Establishment of a highly efficient conjugation protocol for Streptomyces kanamyceticus ATCC12853

et al. 2018

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Kanamycin B as the secondary metabolite of wild‐type Streptomyces kanamyceticus ( S. kanamyceticus ) ATCC12853 is often used for the synthesis of dibekacin and arbekacin. To construct the strain has the ability for kanamycin B production; the pSET152 derivatives from Escherichia coli ET12567 were introduced to S. kanamyceticus by intergeneric conjugal transfer. In this study, we established a reliable genetic manipulation system for S. kanamyceticus . The key factors of conjugal transfer were evaluated, including donor‐to‐recipient ratio, heat‐shock, and the overlaying time of antibiotics. When spores were used as recipient, the optimal conjugation frequency was up to 6.7 × 10 −6 . And mycelia were used as an alternative recipient for conjugation instead of spores; the most suitable donor‐to‐recipient ratio is 1:1 (10 7 :10 7 ). After incubated for only 10–12 hr and overlaid with antibiotics subsequently, the conjugation frequency can reach to 6.2 × 10 −5 which is sufficient for gene knockout and other genetic operation. Based on the optimized conjugal transfer condition, kanJ was knocked out successfully. The kanamycin B yield of kanJ ‐disruption strain can reach to 543.18 ± 42 mg/L while the kanamycin B yield of wild‐type strain was only 46.57 ± 12 mg/L. The current work helps improve the content of kanamycin B in the fermentation broth of S. kanamyceticus effectively to ensure the supply for the synthesis of several critical semisynthetic antibiotics.

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“…The ATP glk and PP glk null mutants were constructed from the parental strain S. peucetius var. caesius as recently described (Rocha et al, 2018 ). These mutants are available at the UNAM-48/WFCC (Mexico City).…”

Section: Methodsmentioning

confidence: 99%

Dissecting the role of the two Streptomyces peucetius var. caesius glucokinases in the sensitivity to carbon catabolite repression

Rocha-Mendoza

Manzo-Ruiz

Romero-Rodríguez

et al. 2021

Journal of Industrial Microbiology and Biotechnology

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Streptomyces peucetius var. caesius, the doxorubicin-producing strain, has two glucokinases (Glk) for glucose phosphorylation. One of them (ATP-Glk) uses adenosine triphosphate as its phosphate source, and the other one uses polyphosphate (PP). Glk regulates the carbon catabolite repression (CCR) process, as well as glucose utilization. However, in the streptomycetes, the specific role of each one of the Glks in these processes is unknown. With the use of PP- and ATP-Glk null mutants, we aimed to establish their respective role in glucose metabolism and their possible implication in the CCR. Our results supported that in S. peucetius var. caesius, both Glks allowed this strain to grow in different glucose concentrations. PP-Glk seems to be the main enzyme for glucose metabolism, and ATP-Glk is the only one involved in the CCR process affecting the levels of α-amylase and anthracycline production. Besides, analysis of Glk activities in the parental strain and the mutants revealed ATP-Glk as an enzyme negatively affected by high glucose concentrations. Although ATP-Glk utilizes only ATP as the substrate for glucose phosphorylation, probably PP-Glk can use either ATP or polyphosphate. Finally, a possible connection between both Glks may exist from the regulatory point of view.

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Development of an efficient conjugal DNA transfer system between Escherichia coli and a non-sporulating Streptomyces strain

Cited by 7 publications

References 34 publications

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

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

Establishment of a highly efficient conjugation protocol for Streptomyces kanamyceticus ATCC12853

Dissecting the role of the two Streptomyces peucetius var. caesius glucokinases in the sensitivity to carbon catabolite repression

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