Heterologous Expression of PrtH a Cell Envelope Proteinase of [i]Lactobacillus helveticus[/i] in [i]Streptococcus thermophilus[/i] LMD-9
Résumé
Streptococcus thermophilus and Lactobacillus helveticus are lactic acid bacteria (LAB) used in fermented dairy products as co-starters. Their growth depends on the supply of amino acids by a proteolytic system, which contains cell envelope proteinases (CEP) hydrolyzing milk proteins in peptides, peptide transporters and intracellular peptidases hydrolyzing peptides into amino acids. Whereas most LAB possess one CEP, L. helveticus strains can display one to four CEP genes. In L. helveticus CNRZ32, the four PrtH, PrtH2, PrtH3 and PrtH4 are potentially present on the cell wall and participate to the generation of techno- and bio-functional peptides. To date, activity and specificity of each CEP is still not established although it is an industrial challenge for strain selections. As biochemical and genetical strategies have been ineffective to study each CEP, we decided to express CEP on another LAB surface. Our strategy was to express PrtH on the cell wall of S. thermophilus LMD¬9. This strain was chosen because it grows under the same culture conditions as L. helveticus, expresses a unique CEP, PrtS, and is naturally transformable. Three mutants were obtained by allelic replacement of prtS: i) a negative control PrtS- with prtS deleted, ii) a PrtH+ mutant expressing PrtH, using the lactobacilli S-Layer motif as anchoring domain and iii) a PrtH+WANS mutant expressing PrtH fused with PrtS cell-wall spacer and anchor domains. All genes were expressed as shown by RT-qPCR. However, only PrtH+WANS proteinase was detected by shaving of the cell-wall proteins and peptide identification by chromatography coupled on line with ESI orbitrap tandem mass spectrometry. These first results show that S. thermophilus is a good tool to secrete and anchor heterologous proteins on the cell wall. Next step is to express the three others L. helveticus proteinases and to determine their catalytic activity on milk proteins.