Molecular cloning and expression of the Cellulomonas flavigena cell-associated amylase gene in E. coli and Cellulomonas flavigena

Abstract

The gene encoding the inducible cell-associated amylase activity was cloned on a 1.5kb Pst1 fragment into pUC8 in E.coli giving the recombinant plasmid pJA 871, and subcloned onto a shuttle vector, pJA85, and transferred into Cellulomonas flavigena AP1(amy-). Expression was observed in both organisms with increased levels being observed from the recombinant in Cellulomonas compared to the parent strain. The 1.5kb fragment was reoriented in pJA871 and the same level of expression observed in both orientations. Tn1000 insertions into the cloned fragment revealed the location of the coding region. Nucleotide sequencing of both ends of the cloned fragment revealed one open reading frame preceded by a putative control region.