For the purpose of $\underline{Bacillus}$ $\underline{subtilis}$ as host cells for genetically engineered genes to over-produce useful products extracellularly, promoters which function strongly in $\underline{B.}$ $\underline{subtilis}$ were tried to develop in this study. The native promoter of an endoglucanase gene of $\underline{B.}$ $\underline{subtilis}$ origin was removed and the remaining structural gene(ENG) was used as a model. To the ENG, strong promoters such as tac promoter (Ptac) and promoters isolated from $\underline{B.}$ $\underline{subtilis}$ 168 were fused and their functions in $\underline{B.}$ $\underline{subtilis}$ were observed. These promoters were then modified by altering nucleotide sequences and their responses on transcriptional efficiency were analyzed to find general requirements on the structure of a strong promoter in $\underline{B.}$ $\underline{subtilis}$.
The ENG structural gene contained in pBSU1028 was isolated and fused to Ptac of pKK223-3 to make a new endoglucanase gene Ptac-ENG in which the native promoter of $\underline{B.}$ $\underline{subtilis}$ endoglucanase gene was replaced by Ptac. The newly constructed plasmid Ptac-ENG is designated pBSKTA. With pBSKTA, $\underline{E.}$ $\underline{coli}$ JM103 was transformed to make a new transformat $\underline{E.}$ $\underline{coli}$ JM103 (pBSKTA). The new endoglucanase gene under control of Ptac in $\underline{E.}$ $\underline{coli}$ JM103 (pBskta) expressed very well in LB medium when induced by IPTG and produced endoglucanase up to 23\% of the total cell protein. However, most of the endoglucanase produced was remained in the cell as inclusion body having no enzyme activity. The Ptac-ENG gene in pBSKTA was transferred to pUB110, a $\underline{Bacillus}$ vector, and with the resulting plasmid pBSKTAU, $\underline{B.}$ $\underline{subtilis}$ RM125 was transformed to make a new transformant $\underline{B.}$ $\underline{subtilis}$ RM125 (pBSKTAU). The Ptac-ENG gene expressed in $\underline{B.}$ $\underline{subtilis}$ RM125 (pBSKTAU) and produced endoglucanase indicating that the Ptac originally developed to use in $\underline{E.}$ $\underline{coli}$ can function in $\underline{B.}$ $\underline{subtilis}$ also.
The production of endoglucanase by $\underline{B.}$ $\underline{subtilis}$ RM125 (pBSKTAU) was however, one third of that by $\underline{B.}$ $\underline{subtilis}$ RM125(pCK98) at the peak. The plasmid pCK98 contains ENG structural gene with native promoter of $\underline{B.}$ $\underline{subtilis}$ origin, whereas plasmid pBSKTAU has ENG with $\underline{E.}$ $\underline{coli}$ oriented Ptac. Therefore, it may be concluded that the strong function of Ptac known in $\underline{E.}$ $\underline{coli}$ cannot be expected in $\underline{B.}$ $\underline{subtilis}$. The promoter Ptac has space length of 16 base pairs between -35 and -10 region, whereas that of endoglucanase gene native promoter is 17 base pairs. Assuming such space length difference makes functional difference, the space length of Ptac was modified to have 17, 18, and 19 base pairs. The promoters having different space lengths such as tac(16 bp), trc(17 bp), toc(18 bp), and tuc(19 bp) were fused to ENG and four new genes, PtacCMC, PtrcCMC, PtocCMC and PtucCMC were constructed. With these genes $\underline{B.}$ $\underline{subtilis}$ RM125 was transformed and functional efficiency of the four promoters on production of endoglucanase were compared. The efficiency ratio between Ptac:Ptrc:Ptoc:Ptuc was 100:101:120:151, suggesting that the space length difference between -35 and -10 region is not responsible for the weak function of Ptac in $\underline{B.}$ $\underline{subtilis}$.
Promoters were cloned from $\underline{B.}$ $\underline{subtilis}$ 168 genome in $\underline{E.}$ $\underline{coli}$ using a promoter probe vector p602/8 which contain chloramphenicol resistance gene(CAT) without promoter. About 80 promoters were cloned and divided into three groups based on their activity; strong, middle, and weak. Some promoters belonging to the strong group functioned very well and produced CAT protein up to 30\% of the total cell protein in $\underline{B.}$ $\underline{subtilis}$ when fused to the CAT gene. The three groups of $\underline{B.}$ $\underline{subtilis}$ promoters cloned were analyzed for their nucleotide sequences and transcription start points to find any consensus sequences related to their promoter efficiencies. All promoters belonging to the strong group showed high conservation of A and T at positions -12 and -11 in the -10 region. However, none of the strong promoters had their nucleotide sequences which agree perfectly to the known consensus sequences.
대장균에서 섬유소분해 효소 유전자를 대량생산하기 위하여 발현용 프로모터인 tac 프로모터와 그 유도체 프로모터인 trc, toc, 그리고 tuc 프로모터를 이용하여 대장균에서 총세포 단백질의 20%수준까지 대량생산을 이루었으나 대부분이 생물학적 활성을 잃은 inclusion body 형태로 판명되어 실질적인 생산정도는 약했다. 이에 대한 대안으로 Bacillus subtilis 를 숙주세포로 선택했다.
Bacillus subtilis 에서의 대량생산을 위해서는 강력한 전사신호가 필수적이나 프로모터 구조에 대한 뚜렷한 연구결과가 미비 했으므로 우선 대장균 프로모터인 tac 프로모터를 B. subtilis 에서의 이용여부를 조사해본 결과, B. subtilis의 성정과 동시에 tac 프로모터는 효율적으로 전사됨을 알았고, 프로모터 탐지용 운반체인 p60218에 옮겼을 때 약 3.6%의 총세포 단백질양에 해당하는 CAT 단백질을 합성했다.
그러나 대장균에서 처럼 강력하지는 않았으므로, B. subtilis 168 염색체에서 강력한 프로모터를 선별하여 구조를 분석하고 또 대량생산에 이용하고자 했다. 이러한 목적으로 B. subtilis 168 염색체에서, 프로모터 탐지용 운반체인 p60218을 이용하여 83 클론을 대장균과 B. subtilis로 클로닝 했다.
이 프로모터들은 B. subtilis에서 31 mU에서 5000 mU의 CAT 활성을 보여 주었고 BJ 2, BJ 23, BJ 25, BJ 27, BJ 29, BJ 31, BJ 38, BJ 65 프로모터는 B. subtilis 에서 총세포 단백질의 20 - 30%에 해당하는 CAT 단백질 합성을 보여주는 강력한 프로모터로 판명 되었다.
이들 프로모터를 섬유소 분해효소 유전자의 상류부위에 연결시켜 섬유소 분해효소 활성을 약 6 U/ml 수준까지 올렸으며 섬유소 분해효소는 모두 균체 밖으로 분비됨을 확인 했다.
DNA 염기서열 분석과 S1 mapping 실험을 통하여 강력한 프로모터의 구조와 전사개시점을 알아냈으며 그결과는 기존에 보고되었던 결과들과 매우 달랐다. -35 상류부위의 A 밀집 혹은 AT 다발염기서열이 존재하지 않았으며, -16, -15 위치의 TG 염기의 보존도 존재하지 않았다. 또한 -10 부위의 consensus sequence의 염기보존성 패턴이 크게 다름을 발견할 수 있었으며, 뚜렷한 특징으로 일렬로 늘어선 중복형의 프로모터 구조를 대부분의 강한 프로모터가 갖고 있음을 발견했다. 이러한 특징적인 구조가 강력한 전사신호에 관련이 될 것이라고 제안 했으며 이에대한 실험적 타당성은 앞으로의 실험에서 밝혀 질 것이다.
