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Dna sequence of genes involved in tyramine production

An important bottleneck in the study of BA producing pathways at the beginning of the project was that (excepted hdcA) the structural genes coding for the decarboxilases and the transporters had not been identified. Cloning, molecular analysis, and genetic engineering of the genes involved in BA production in wine and fermented milk was performed to achieve the objectives.

Sequencing of the tyrosine decarboxylase cluster of Enterococcus durans IPLA 655. Two degenerate primers were designed, based on the partial sequence of the tyrosine decarboxylase protein from Lb. brevis IOEB 9809. These were used to test E. durans IPLA 655 by PCR for the presence of the tyrosine decarboxilase gene. The obtained 820 bp amplicon was cloned and sequenced.

The sequence analysis revealed an ORF which showed strong similarity to the tyrosine decarboxylase genes of E. faecalis JH2-2 (83% identity) and Lb. brevis IOEB 9809 (73% identity). This gene was designated tdcA. Using reverse PCR, the E. durans genome was walked on either side of this fragment. Upstream of tdcA, a complete ORF was found that shared 78% and 72% identity respectively with the tyrosyl t-RNA syntethases of E. faecalis JH2-2 and Lb. brevis IOEB 9809.

This gene was designated tyrS. The analysis of its sequence revealed that the encoded tyrosyl-tRNA synthetase (TyrS) belongs to the class I aminoacyl-tRNA synthetases, characterized by HIGH and KMSKS motifs. The HIGH motif is perfectly conserved in E. durans IPLA 655 TyrS and the KMSKS motif is represented by the KFGKT sequence, as in E. coli, Bacillus subtilis, and E. faecalis.

Downstream of tdcA, another ORF (tyrP) was found that shared 84% and 66% identity respectively with the antiporters of E. faecalis JH2-2 and Lb. brevis IOEB 9809. The deduced amino acid sequence of this protein (determined using the Sosui program) revealed a membrane protein structure with 11 transmembrane helices. This protein may be involved in tyrosine/tyramine exchange.

Sequencing of the tyramine producing pathway of the wine LAB Lactobacillus brevis IOEB 9809:A strategy based on linker-mediated PCR was employed to sequence the L. brevis IOEB 9809 genomic sequence surrounding the 792 bp-fragment of tyrDC determined in a previous work. A 7979 bp DNA sequence was determined. It contains four complete and one partial open reading frames (ORFs) encoding polypeptides larger than 100 amino acids.

The tyrDC gene codes for a protein of 626 amino-acids with a calculated molecular weight of 70.5 kDa in agreement with experimental results. The upstream ORF showed strong similarities with genes of tyrosyl-tRNA synthetase (tyrRS).

Downstream of tyrDC were two ORFs related to genes of amino-acid permeases (tyrP) and Na+/H+ antiporters (nhaC), respectively, and the 5 -end of a fifth ORF similar to ornithine transcarbamylase genes (otc).

Sequencing of the tyramine producing pathway of food LAB Carnobacterium divergens AN 508: To determine the presence of a tyrDC gene amonst known tyramine producing carnobacteria, PCRs were carried out using the degenerated primers P1-rev and P2-for (Lucas and Lonvaud-Funel, 2002).

An amplification product of ~ 800 base pairs was obtained with the strains C. divergens AN 508 and C. piscicola AN 545. The PCR product for the two carnobacteria were sequenced (78.7% sequence similarity). They showed also high similarities with the published sequenced of Lb. brevis IOEB 9809 and Enterococcus faecalis.

The complete tyrDC gene sequence of C. divergens was determined by use of inverse and conventional PCR. A 2.2kb PCR product was obtained and sequenced. It was located at the 3-end of the previously sequenced fragment of the tyrDC gene. To obtain the 5-end of this gene, a degenerated primer was designed on the basis of a comparison of the sequence of tyrRS genes identified in E. faecalis and other LAB because sequencing of the tyrDC cluster of E. faecalis had previously suggested the presence a tyrRS gene upstream of tyrDC .

A 900-bp DNA fragment was successfully amplified using this strategy. After sequencing, it was confirmed that it contained the 5-end of the tyrDC gene and a fragment of a tyrRS gene. The complete sequence of the tyrDC cluster determined in C. divergens AN 508 comprises 3427 bp including the three genes tyrRS, tyrDC and tyrP.

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