CLONING OF A DNA FRAGMENT CARRYING THE 4-HYDROXYCINNAMATE DECARBOXYLASE (POFK) GENE FROM KLEBSIELLA-OXYTOCA, AND ITS CONSTITUTIVE EXPRESSION IN ESCHERICHIA-COLI JM109 CELLS

The 4-hydroxycinnamate decarboxylase gene (pofK gene) was cloned from Klebsiella oxytoca, an epiphytic bacterium able to decarboxylate hydro xycinnamic acids to styrene derivatives, in Escherichia coli JM109. Co lonies of the enzyme activity-positive transformants were screened by a selection assay combined with an antifungal test using Cladosporirnn herbarum IHU9262 as the bio-indicator. Two positive transformants con stitutively producing 4-hydroxycinnamate decarboxylase were obtained. One of the transformants had a recombinant plasmid designated as pTCD1 00 in which a 9.6kb HindIII segment carrying pofK gene was contained. As the expression of the pofK gene in K. oxytoca was substrate-inducib le, it was most likely that the pofK gene expression in E. coli cells was free from any regulation by a pofK gene repressor. postulated in K . oxytoca cells. The decarboxylase synthesized in E. coli cells showed almost the same specific activity as that of K. oxytoca induced by th e substrate.