Construction of a two - cistron system encoding endoglucanase and β - glucosidase to be expressed in escherichia coli and saccharomyces cerevisiae

Abstract

Construction of a two-cistron systim comprising an endoglucanase gene and a $\beta$-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 $\beta$-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 tactransferred from plasmid pKK223-3. A Shine-Delgarno (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-$\beta$-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 $\beta$-glucosidase at the levels of 15\% and 22\% of the total cell protein, respectively. The production of $\beta$-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 $\beta$-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 $\beta$-glucosidase was produced when the distance between the two genes was 17 base pairs, but no $\beta$-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 $\beta$-glucosi...