A pSET152 derivative vector marked with thiostrepton resistance gene for introducing DNA into Streptomyces

Abstract:

[Objective] In the PCR-targeting system of Streptomyces, apramycin is the most widely used selection marker. However, using apramycin as selection marker at disruption stage precludes, during subsequent genetic complementation, the use of some very useful and widely used vectors with the same marker, such as pSET152. This often caused unwanted inconvenience, especially in the situation that the physiological function of interested gene is sensitive to gene dosage, such as regulatory genes. The current study aims to provide an integrative plasmid with selection marker rather than apramycin. [Methods] Fusion-PCR and λ Red recombination were adapted to construct the new vector. [Results] The bla gene from pCR2.1 and the tsr gene from pHZ1358 were fused in the tsr-bla order. This fused fragment replaced the aac(3)-IV gene on pSET152 to generate pGIM6626. This new vector was validated by successfully restoring granaticin production of a granaticin-deficient S. vietnamensis mutant by re-introducing the deleted minimal polyketide synthase genes. [Conclusion] We constructed a new pSET152 derivative vector, pGIM6626, which contains ampicillin and thiostrepton resistance genes for selection in E. coli and Streptomyces, respectively. pGIM6626 and pSET152 have similar uses, but the former is more compatible with the PCR-targeting system because of no conflict of selection marker.