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 Cladosporium 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 9.6 kb 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.
a JRDC Plant Ecochemicals Project, RBP Center Bldg. , 1-1 Megumino Kita 3-chome, Eniwa-shi, Hokkaido 061-13 , Japan.
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Hashidoko
a JRDC Plant Ecochemicals Project, RBP Center Bldg. , 1-1 Megumino Kita 3-chome, Eniwa-shi, Hokkaido 061-13 , Japan.
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Urashima
a JRDC Plant Ecochemicals Project, RBP Center Bldg. , 1-1 Megumino Kita 3-chome, Eniwa-shi, Hokkaido 061-13 , Japan.
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Mizutani