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 hydroxycinnamic acids to styrene derivatives, in Escherichia coli JM109. Colonies 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 constitutively producing 4-hydroxycinnamate decarboxylase were obtained. One of the transformants had a recombinant plasmid designated as pTCD100 in which a 9.6kb HindIII segment carrying pofK gene was contained. As the expression of the pofK gene in K. oxytoca was substrate-inducible, 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 the substrate.