Development of expression system for cellulase genes in bacillus subtilis

Abstract

Development of an effective expression system for foreign genes in Bacillus subtilis was attempted. A strong promoter, BJ27, was screened from chromosome of B. subtilis 168 using a promoter probe vector, p602/8. The nucleotide sequence of the BJ27 promoter was analyzed and its transcription initiation site was determined. The promoter contained the classical sequence to be recognized by $\sigma^{43}$. The native promoter of the B. subtilis endoglucanase gene contained in pCK98 was removed and replaced by the BJ27 promoter. The resulted plasmid was designated as pJH27E. With this plasmid, B. subtilis was transformed. The transformant B. subtilis(pJH27E) produced endoglucanase with activity three times higher than that produced by B. subtilis (pCK98). To improve translation efficiency, a synthetic Shine-Dalgarno sequence which perfectly matched with the 3`` $^\prime$ end of the 16S rRNA of B. subtilis, or the SD sequence of $\phi$ 10 gene of T7 phage, were integrated into the endoglucanase gene. It was found that the synthetic SD was more effective than the SD of T7 phage $\phi$ 10 gene on the translation of the endoglucanase gene. Further increase of expression efficiency was obtained by integration of an AT rich sequence into BJ27 at upstream of the -35 region together with deletion of 88 base pairs between the transcription initiation site of BJ27 and the translation initiation site that has a strong secondary structure. The B. subtilis strain harboring the reconstructed gene produced endoglucanase having 20-fold higher activity compared to the strain carrying the unmodified gene. Most of the endoglucanase produced was secreted into the culture medium and occupied up to 60% of the total extracellular protein. A simultaneous production of endoglucanase and β-glucosidase was possible by constructing two cistron systems in which the endoglucanase is ahead of the β-glucosidase gene and vice versa. When SD sequence was inserted between the two genes, the two ...