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 secodary 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 enzymes were produced as much as the single gene did. When the β-glucosidase gene was fused to the endoglucanase gene, a hybrid protein was produced and secreted. The hybrid protein showed normal endoglucanase activity but the β-glucosidase activity was reduced significantly.
강력한 promoter들과 효과적인 SD sequence들을 사용해서 섬유소 분해 효소들을 B. subtilis에서 대량으로 생산하는 system을 개발하였다. B. subtilis 에서 작용하는 강력한 promoter들은 promoter probe vector, p602/8 을 이용해서 B. subtilis 168 염색체로부터 분리하였으며, 이중에서 가장 효과적인 BJ27 promoter의 염색체서열과 전사개시점을 분석하였다. 분석결과 BJ27 promoter는 $\sigma^{43}$ 에 의해 인식되는 promoter 였음을 밝혀내었다. 이 promoter 를 이용해서 endoglucanase 유전자를 B. subtilis에서 발현시켰을 때 (pJH27E), endoglucanase 원래의 promoter (pCK98) 에 의해서 생산되는 endoglucanase 활성의 약 3배의 활성을 보였다.
Translation 효율을 개선하기위해, 두가지의 SD sequence를 사용했다. 첫번째 SD는 B. subtilis 의 16S rRNA의 3' 말단부위와 완전히 match 되는 합성 SD sequence 이고, 두번째 SD 로는 E.coli 에서 효과적인 SD 로 알려진 T7 phage 의 $\phi$10 유전자의 SD sequence 를 이용했다. 이 두 SD sequence 를 endoglucanase 유전자에 접목시켰을때, 합성 SD sequence 가 T7 SD sequence 에 비해 endoglucanase 의 translation 에 보다 효과적이었다.
BJ27 promoter 의 -60 부위에 AT-rich sequence 를 첨가시키고, 전사 개시 지점과 번역 개시 지점 사이에 있는 2차구조를 제거시켰을때 (pJH27UD88) endoglucanase 활성이 최고치를 보였다. 생산된 endoglucase는 95% 이상 배지로 분비되었으며, 이렇게 분비된 endoglucanase 는 total extracellular 단백질의 60% 이상을 차지했다. 이 JH27UD88 promoter를 이용해서 β-glucosidase 를 발현시켰을때, 생산된 β-glucosidase 는 B. subtilis 총 단백질의 12% 정도로 발현되었다.
한 Bacillus 세포에서 endoglucanase 와 β-glucosidase 를 동시에 생산하기위해 이 두 유전자를 포함하는 two cistron system 을 개발했다. 두 유전자사이에 SD sequence 를 첨가했을때, 두 효소들의 발현정도는 single cistron system과 거의 같은 수준으로 발현되었다.
