Transformation of Zymomonas anaerobia with a plasmid containing cellulase gene

Abstract

A new buffer system which induce competent state of z.anaerobia as well as E.coli has been developed. Using the new buffer transformation of z.anaerobia with three types of plasmids, i,e, pBR325, pRK250 and pDH73 were succeeded. The main point of the new buffer system is in the use of $Mn^{++}$ ion and lysozyme. The concentration of lysozyme in the buffer was restricted to 0.4 mg/ml which allowed modification of cell wall structure but remained cells alive. Among the three systems developed one system which is relatively simple with high efficiency composed as follows; $MnCl_2$-70mM, 30mM-$CaCl_2$, 10mM-$NaCl$, 100mM-$RbCl_2$, 40mM-$LiCl_2$, 50mM-$CsCl$ and 3mM-$HcoCl_3$. After confirming successful transformation of Z.anaerobia with pBR325 and pRK 2501, another transformation of the same recipient cells with pDH73 was also tried. The plasmid pDH73 which has been constructed in this laboratory contains CMCase gene of B.subtilis. The recipient organism z.anaerobia has been known as an effective ethanol producer. Therefore, the success in transformation of Z.anaerobia with pDH73 in this research will contribute for the future study on ethanol production of Z. anaerpbia using cellulosic materials significantly.