Establishment of a highly efficient conjugation protocol for Streptomyces kanamyceticus ATCC12853

Abstract

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.

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

“…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.…”

“…The conjugation conditions were highly strain-specific and usually ineffective for different actinobacteria. Due to interspecific variation, appropriate conditions for intergeneric conjugation have to be developed and optimized for each particular strain (Sun et al, 2014;Wang and Jin, 2014;Zhang et al, 2018).…”

“…Heat-shock treatment of Streptomyces spores has generally been carried out prior to mixing with donor E. coli (Sun et al, 2014;Zhang et al, 2018). To determine the optimal temperature for heat treatment and incubation time of heat-shocked spores, the effects of temperature and different incubation time on conjugation efficiency were investigated.…”

“…In addition, before the plates are flooded with fresh LB medium containing antibiotics, the incubation time of the mixed culture of Streptomyces and E. coli plays an important role in conjugation efficiency (Zhang et al, 2018). If the time of mixed culture is too short, Streptomyces mycelia grow weakly and are easily scraped off, leading to a lower conjugation efficiency.…”

Trans-Kingdom Conjugation Within Solid Media from <em>Escherichia coli</em> to <em>Saccharomyces cerevisiae</em>

“…Not only can the pipeline generate functional conjugative plasmids-as indicated with the creation and testing of pTA-Mob 2.0-the pipeline can be adapted for use with any desired customizable vector.During the optimization process, it was identified that the ideal holding temperature for the molten agar media, and at the time the cell mixture was added, was 60°C. Previous studies using different bacterial species have also demonstrated that a "heat shock" step during conjugation results in improved conjugation efficiencies[40][41][42]. However, it remains unclear as to why the heat shock at 60°C yields an increased number of transconjugant S. cerevisiae colonies.…”