MOLECULAR CHARACTERIZATION OF AN INDUCIBLE P-COUMARIC ACID DECARBOXYLASE FROM LACTOBACILLUS-PLANTARUM - GENE CLONING, TRANSCRIPTIONAL ANALYSIS, OVEREXPRESSION IN ESCHERICHIA-COLI, PURIFICATION, AND CHARACTERIZATION

By using degenerate primers designed from the first 19 N-terminal amin o acids of Lactobacillus plantarum p-coumaric acid decarboxylase (PDC) , a 56-bp fragment was amplified from L. plantarum in PCRs and used as a probe for screening an L. plantarum genomic bank, Of the 2,880 clon es in the genomic bank, one was isolated by colony hybridization and c ontained a 519-bp open reading frame (pde gene) followed by a putative terminator structure. The pdc gene is expressed on a monocistronic tr anscriptional unit, which is transcribed from promoter sequences homol ogous to Lactococcus promoter sequences, No mRNA from pdc and no PDC a ctivity were detected in uninduced cell extracts, indicating that the expression is transcriptionally regulated by p-coumaric acid, which co rresponds to an activation factor up to 6,000, The pde gene was overex pressed constitutively in Escherichia coli, and the recombinant enzyme was purified and characterized.