An excretion reporter construct, tacIQpar8cex, was assembled by the cloning of the Celldomonas fimi exoglucanase gene, cex, into the TaC expression/excretion plasmid, lacIQpar8. E. coli JMlOl harbouring tacIQpar8cex was shown to produce extracellular exoglucanase (Exg) activity. Derivatives of tacIQpar8cex were constructed for studies of the factors that were important to excretion of Exg in JMlOl. The first derivative engineered using an improved double-stranded DNA mutagenesis protocol was tacIQpargcex/NB, in which a unique Bam HI site had been introduced into a non-essential region of tacIQpar8cex. Quantitatively, tacIQpar8cex and tacIQpargcex/NB expressed virtually the same level of Exg activity in the culture supernatants of their hosts. The tacIQpargcex/NB construct was employed t...[ Read more ]

An excretion reporter construct, tacIQpar8cex, was assembled by the cloning of the Celldomonas fimi exoglucanase gene, cex, into the TaC expression/excretion plasmid, lacIQpar8. E. coli JMlOl harbouring tacIQpar8cex was shown to produce extracellular exoglucanase (Exg) activity. Derivatives of tacIQpar8cex were constructed for studies of the factors that were important to excretion of Exg in JMlOl. The first derivative engineered using an improved double-stranded DNA mutagenesis protocol was tacIQpargcex/NB, in which a unique Bam HI site had been introduced into a non-essential region of tacIQpar8cex. Quantitatively, tacIQpar8cex and tacIQpargcex/NB expressed virtually the same level of Exg activity in the culture supernatants of their hosts. The tacIQpargcex/NB construct was employed to obtain other derivatives using the Unique Site Elimination mutagenesis approach. One such derivative, tacIQpar8cex/ompA, in which 14 codons of the ompA leader had been deleted, was incapable of expressing extracellular Exg in JMlOl. Another derivative, lacIQpar8cex, was constructed by replacing the tat promoter in tacIQpar8cex with the lac promoter. The cell growth, plasmid stability and ability to produce excretory Exg were compared between strains JMlOl(tacIQpar8cex) and JMlOl(lacIQpar8cex). There was no major differences between the two strains, when the inducer, IPTG, was absent. However, when IPTG was present, cells of JMlOl(lacIQpar8cex) were shown to grow faster, produce higher biomass, and exhibit greater plasmid stability and better excretory Exg activity than of JMlOl(tacIQpar8cex). Supernatant samples of JMlOl(lacIQpar8cex) were devoid of detectable activity of an intracellular enzyme, glucose-6-phosphate dehydrogenase, indicating that the detected Exg activity was genuinely excretory in nature. The efficient performance of JMlOl(lacIQpar8cex) for excretory Exg production suggests that the system may be optimized and extended for use in other proteins.