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

Abstract

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.

Enabling Efficient Genetic Manipulations in a Rare Actinomycete Pseudonocardia alni Shahu

“…Previous studies have reported that an appropriate concentration of Mg 2+ in the medium, or a suitable heat-shock treatment of the cells, or an optimal ratio of donor-to-recipient could give improved frequency of conjugation ( Song et al, 2019 ; Zhang et al, 2019 ). We thus individually assessed each of such factors to attain successful conjugation.…”

Identification of a gene from Streptomyces rimosus M527 negatively affecting antibiotic biosynthesis and morphological differentiation

“…Accordingly, significant focus has been placed on developing genetic transformation methods and expression systems [16] or ribosome engineering technology combined with new fermentation conditions to improve rimocidin production [17]. The long-term goal of our research is to better understand regulatory mechanisms involved in rimocidin biosynthesis.…”

“…The gene nsdA sr was placed under the control of promoter ermE * in plasmid pIB139 to create pIB139-nsdA sr (Additional file 3: Figure S3). The plasmid pIB139-nsdA sr was then introduced into S. rimosus M527 by intergeneric conjugation [16], to generate the recombinant strain S. rimosus M527-NA sr resistant to 300 µg/mL apramycin (Additional file 4: Figure S4). The integration of plasmid pIB139-nsdA sr into the chromosome of S. rimosus M527 was verified by PCR (Additional file 5: Figure S5).…”

“…Streptomyces rimosus M527 (China Center for Type Culture Collection (CCTCC) M2013270), which was originally isolated in 2011 from soil, is also known as rimocidin producer [15]. In 2019, a gene transfer system for the strain was developed [16] and the genome of the strain was sequenced (GenBank accession No. GCA_004196335.1).…”

Effects of S-adenosylmethionine on production of secondary metabolites in Streptomyces diastatochromogenes 1628

“…

Streptomyces are famous for their ability to synthesize a large number of bioactive compounds as secondary metabolites containing antibiotics, enzyme inhibitors, and other small molecules with potential physiological activity (Niu et al, 2016;Song et al, 2019;Yin et al, 2019). Secondary metabolites are produced by a multi-step reaction of a primary metabolite as a precursor (Liu et al, 2013;Li et al, 2021).

…”