Electroporation-mediated transfer and expression of foreign gene in lactobacillus acidophilus

Abstract

The transformation of lactic acid bacteria via electroporation with plasmid DNA and expression of the foreign gene in the new host cells were attempted for the purpose of improving lactic cultures using recombinant DNA methods. $\underline{Lactobacillus}$ $\underline{acidophilus}$ was used as host cells and an endoglucanase gene of $\underline{Bacillus}$ $\underline{subtilis}$ origin was used as a model gene. $\underline{L}$. $\underline{acidophilus}$ cells were grown in MRS broth and cell suspensions of $2\times 10^8$ to $8\times 10^8$ bacteria per ml were prepared in PEB buffer. Into the cell suspensions plasmids pACM1, pACM2, pBSKTAU, and pCK98 containing the endoglucanase gene were added and electroporation was performed. The cells harvested early in the logarithmic growth stage showed the highest transformation frequency. The frequency of transformation was proportional to the field strength of the electroporation pulse at a capacitance of 25uF. Under the optimal conditions the $\underline{L}$. $\underline{acidophilus}$ cells were transformed with frequency form $10^1$ to $10^4$ transformants per ug of DNA. The endoglucanase gene of $\underline{B}$. $\underline{subtilis}$ origin expressed well in $\underline{L}$. $\underline{acidophilus}$, and most of the gene product was found in the culture medium. The efficiency of the endoglucanase production by the transformed $\underline{L}$. $\underline{acidophilus}$ cells depended greatly on the choice of the vector plasmids on which the gene to be inserted. Under the same cultural conditions, the $\underline{B}$. $\underline{subtilis}$ endoglucanase gene in $\underline{L}$. $\underline{acidophilus}$ produced more amount of endoglucanase than in $\underline{B}$. $\underline{subtilis}$ RM125, suggesting that lactic acid bacteria are good candidates for mass production of endoglucanase. The endoglucanase genes contained in pACM1 and pACM2 were found to use their native promoters for expression, because the $\unde...