The conjugation protocols in myxobacterium Sorangium cellulosum are often inapplicable due to the strain-specific sensitivity to the presence of Escherichia coli cells or the resistances to many antibiotics. Here we report that the conjugative transfer of the mobilizable plasmid pCVD442 from E. coli DH5alpha (lambda pir) to Sorangium strains could be greatly increased by the presence of low doses of dual selection antibiotics in the mating medium. The improvement was efficient in either E. coli-tolerant or sensitive Sorangium strains. For those phleomycin and hygromycin tolerant Sorangium strains, chloramphenicol-resistance gene was developed as a new selectable marker by driving the resistance gene with the aphII promoter. Using the improved protocol, the epothilone biosynthetic pathway was inactivated by an insertion mutation in the biosynthetic genes of the producing Sorangium strains.