Application of a replicative targetable vector system for difficult-to-manipulate streptomycetes

The low frequency of homologous recombination together with poor efficiency in introducing DNA into the cell are the main factors hampering genetic manipulation of some bacterial strains. We faced this problem when trying to construct mutants of Streptomyces iranensis DSM 41954, a strain in which conjugation is particularly inefficient, and suicidal vectors had failed to yield any exconjugants. In this work, we report the construction and application of a conjugative replicative vector, pDS0007, which allows selection of exconjugants even with poor conjugation efficiency. The persistence of the construct inside the cell for as long as required facilitates the homologous recombination events leading to single and double crossovers. While it was confirmed that the vector is frequently lost without selection, the recognition sequence for the I-SceI endonuclease was included in the backbone of pDS0007. The presence of a I-SceI recognition sequence would allow to force the loss of the vector and the appearance of double crossover recombinants by introducing a second construct (e.g. pIJ12742) that expresses a Streptomyces codon-optimised gene encoding the I-SceI endonuclease. To facilitate screening for vector-free clones, the construct also carries a Streptomyces codon-optimised gusA gene encoding the β-glucuronidase expressed from a constitutive promoter. We prove the usefulness of this vector and strategy with the strain S. iranensis DSM 41954, in which we could readily delete an essential gene of a newly discovered biosynthetic pathway for a phosphonate-containing natural product, which led to loss of phosphonate production according to ³¹P NMR spectroscopy. KEY POINTS: • pDS0007 is a new vector for gene-targeting in difficult-to-manipulate streptomycetes. • pDS0007 is self-replicative but easy to cure, targetable and allows visual screening. • pDS0007 was used to prove the discovery of a novel phosphonate biosynthetic pathway.