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A recombinant Lactococcus lactis strain expressing the tyrosine decarboxylation pathway

Biogenic amines (BA) are the product of decarboxylation pathways in food bacteria and a major cause of food poisoning. Substrates of decarboxylation pathways are amino acids that are converted into the corresponding amines or amino acids (e.g. histidine/histamine, aspartate/alanine or tyrosine/tyramine).

The pathways consist of a transporter that catalyses the translocation of the substrate into the cell coupled to the secretion of the metabolic end product of the same substrate out of the cell, and a decarboxylase residing in the cytoplasm. In order to prevent the formation of BA by the organisms, it is essential to understand the physiological relevance of the pathways for the organisms.

The benefit of the pathway for the microorganisms may be in the generation of metabolic energy and/or acid stress resistance. The pathways function as indirect proton pumps and, therefore, generate proton motive force. The two components of the proton motive force are generated in the two separate steps of the pathway. The transporter exchanges two substrate that differ in charge, which results in membrane potential, and the decarboxylation steps consumes a cytoplasmic proton, which results in a pH gradient across the membrane.

The alkalinising effect of the pathway is also responsible for the acid stress function. While the different properties of the enzymes in the pathway have been established in vitro, the physiological benefit can only be studied in vivo by studying the performance of the pathway as a whole. For this purpose we have constructed a recombinant Lactococcus lactis strain containing a synthetic operon encoding for a tyrosine decarboxylation pathway.

A synthetic operon was constructed containing the tyrosine decarboxylase tyrDC and the tyrosine/tyramine exchanger tyrP in the L. lactis expression vector pNZ8048 under control of the nisin promoter. The two genes originated from Lactobacillus brevis. The tyrDC gene was placed upstream of the tyrP gene and was tagged with a His-tag. Expression of the TyrDC protein was demonstrated by immunoblotting using antibodies against the His-tag. Expression of the TyrP protein was demonstrated by preparing right-side-out membranes and measuring tyrosine/tyramine exchange activity. Two assays were developed to assay for tyrosine decarboxylase activity of the cells.

In one assay, radioactive tyrosine labelled at the C1 position was added to the cells. The C1 atom of tyrosine is released from the molecule as carbon dioxide upon decarboxylation. As carbon dioxide is easily released from the suspension, decarboxylase activity may be monitored from the decrease of the radioactivity from the suspension. Samples are taken in time and the radioactivity is measured in a liquid scintillation counter.

The assay is simple and fast but requires significant conversion. In the second assay, the cell suspension is incubated with uniformly labeled tyrosine. Samples are taken in time and spotted on a TLC plate that is developed in a mixture of propanol, butanol and water. Radioactive spots on the plates are visualized using a phosphoimager.

The assay is time consuming, but allows for a good monitoring of the purity of the substrate and the products developed. The tyrosine decarboxylation activity of L. lactis cells expressing the pathway was about half the activity of cells expressing the tyrosine decarboxylase alone.

L. lactis cells producing the TyrDC protein showed essentially no tyrosine decarboxylation activity suggesting that the produced protein was inactive. In agreement, fractionation of the cells followed by detection of the protein in the different fractions demonstrated that all protein was present as inclusion bodies. Several approaches were followed to reduce the formation of inclusion bodies in order to improve the decarboxylation activity of the cells.

The approaches included optimising the inducer concentration, changes to the composition of growth medium, lower temperature during growth and a lower initial pH. Several conditions independently resulted in significant improvement of the activity of the cells: lowering of the growth temperature to 25C, lowering of the initial pH of the growth medium to 6, addition of KCl to the growth medium, and addition of compatible solutes.

The common factor in these conditions is stress. The activity of the cells could be further improved by combining the different conditions. Ultimately, a cell suspension at an optical density of 50 could convert 1mM of tyrosine in 24 h.

Contacto

Julius LOLKEMA, (Group leader)
Tel.: +31-50-3632155
Fax: +31-50-3632154
Correo electrónico
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