Abstract: The aim of this study was to construct new generation shuttle vectors harboring bacteriophage T4 lysozyme gene (gene e). T4 lysozyme gene was inserted into pMK3 and pNW33N shuttle vectors of Escherichia coli-Bacillus sp. to construct pMK3L and pNW33NL, respectively. pMK3L and pNW33NL recombinant plasmids were then transferred into Bacillus subtilis by electrotansformation. Escherichia coli-Saccharomyces cerevisiae shuttle vector bearing gene e, pRS416L, constructed in the previous study was also used for directly transformation into Saccharomyces cerevisiae in this study. Escherichia coli plasmid vectors bearing gene e could facilitate the isolation of plasmid DNA without using cell wall and cell membrane disruptive agents such as lysozyme and Etyhlene Diamine Tetra Acetic Acid (EDTA). Therefore, these lysates prepared by using Tris-EDTA (TE) buffer or distilled water of Escherichia coli including gene e could directly be used for transformation into target Bacillus subtilis or Saccharomyces cerevisiae without plasmid purification procedures.