Construction of a two-cistron system comprising an endoglucanase gene and a β-glucosidase gene was attempted to produce efficiently the corresponding cellulases using Escherichia coli and Saccharomyces cerevisiae as host cells. The endoglucanase gene from Bacillus subtilis and β-glucosidase gene from Cellulomonas fimi previously cloned in our laboratory were used. The two genes were so arranged as to be controlled under a strong promoter tac transferred from plasmid pKK223-3. A Shine-Dalgarno (SD) sequence from plasmid pET3a was inserted between the two genes to increase translation efficiency of the second cistron. These components in the constructed cistrons are thus lined in the order of ptac-endoglucanase gene-SD-β-glucosidase gene.
With the two cistron genes, E. coli strains were transformed and the expression in L. medium and by inducing with IPTG. Both genes expressed well and produced endoglucanase and β-glucosidase at the levels of 15 % and 22 % of the total cell protein, respectively. The production of β-glucosidase, product of the second cistron after the SD sequence, may be associated by a coupled effect of the upstream translation. When the space between the termination codon of the first cistron and the initiation codon of the second cistron was increased from 50 base pairs to 1055 base pairs, the 22 % production of β-glucosidase was reduced to 14 %. The coupled translation in the two-cistron system was further supported by the fact that even without the SD sequence for the second cistron some amount of β-glucosidase was produced when the distance between the two genes was 17 base pairs, but no β-glucosidase was produced when the second gene was separated from the first gene by 1022 base pairs.
Saccharomyces cerevisiae cells were transformed with the two cistron system and were grown in YPD medium. Both genes expressed in the yeast and produced endoglucanase of 2,427 mU/ml activity and β-glucosidase of 16 U/ml.
대장균과 효모에서 섬유소 분해효소를 효율적으로 발현시키기 위하여 endoglucanase와 β-glucosidase 유전자를 포함하는 Two-cistron을 제작하였다. Endoglucanase 유전자는 Bacillus subtilis 로 부터 그리고 β-glucosidase 유전자는 Cellulomonas fimi로 부터 이미 우리 실험실에서 cloning 된 유전자를 사용하였다. Promoter는 plasmid pKK223-3의 tac promoter를 사용하였다. Plasmid pET3a 의 SD sequence를 second cistron 의 translational efficiency를 높히기 위하여 두 유전자 사이에 넣었다. 이 요소들은 ptac-endoglucanase gene-SD-β-glucosidase gene 순서로 제작되었다.
이 two cistrons를 대장균에 transformation하고 IPTG로 induction하여 두 유전자의 발현 정도를 조사하였다. Endoglucanase는 총 세포 단백질의 15 % 그리고 β-glucosidase는 22 % 까지 발현되었다. β-Glucosidase의 생산은 upstream translation에 의한 coupled effect가 존재하는 것으로 생각되었다. First cistron의 termination codon과 second cistron의 initiation codon의 간격을 50bp에서 1,055로 늘렸을 경우 22 %에서 14 %로 감소하였다. 또한 SD sequence가 없는 plasmid pTC17의 간격을 17 bp에서 1022 bp로 늘렸을 경우 β-glucosidase의 효소 활성도는 사라져 버렸다.
Two-cistron system을 효모에 transformation시켜 효소 활성도를 측정한 결과 endoglucanase는 2,427 mU/ml 그리고 β-glucosidase는 16 U/ml의 효소 활성도를 보여 주었다.
