Heterologous expression of the plant coumarate:CoA ligase in Lactococcus lactis

Aims: To demonstrate the expression of coumarate : CoA ligase of Arabidopsis thaliana in Lactococcus lactis as a first step of cloning the vanillin pathway. Methods and Results: The 4CL gene was amplified from a cDNA library of A. thaliana by PCR and subcloned into a multicopy lactococcal vector where the expression is under the nisA promoter. The maximum yield of the protein in the recombinant strain of L. lactis was obtained 3 h after induction with 10 ng ml(-1) of nisin. However, these levels were only fraction of those detected in cell extracts of Pseudomonas fluorescens AN103 strain which naturally expresses its own enzyme when grown in the presence of ferulic acid as a carbon source. Among different substrates examined, the enzyme was most active against coumaric acid. Conclusions: The gene encoding coumarate : CoA ligase in A. thaliana was isolated, sequenced, cloned and expressed in L. lactis. Significance and Impact of the Study: This study represents the first of the two steps for genetic engineering of the vanillin pathway in the GRAS (generally recognized as safe) organism L. lactis.