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Background  From fundamental studies to industrial processes, synthesis of heterologous protein by micro-organisms is widely employed. The secretion of soluble heterologous proteins in the extracellular medium facilitates their recovery, while their attachment to the cell surface permits the use of the recombinant host cells as protein or peptide supports. One of the key points to carry out heterologous expression is to choose the appropriate host. We propose to enlarge the panel of heterologous secretion hosts by using  Streptococcus thermophilus  LMD-9. This lactic acid bacterium has a generally recognised as safe status, is widely used in the manufacture of yogurts, fermented milks and cheeses, and is easy to transform by natural competence. This study demonstrates the feasibility of secretion of a heterologous protein anchored to the cell surface by  S. thermophilus . For this, we used the cell envelope proteinase (CEP) PrtH of  Lactobacillus helveticus  CNRZ32 CIRM-BIA 103.    Results  Using  S. thermophilus  LMD-9 as the background host, three recombinant strains were constructed: i) a negative control corresponding to  S. thermophilus  PrtS -  mutant where the  prtS  gene encoding its CEP was partially deleted; ii) a PrtH +  mutant expressing the  L. helveticus  PrtH pro-protein with its own motif (S-layer type) of cell-wall attachment and iii) a PrtH + WANS mutant expressing PrtH pro-protein with the LPXTG anchoring motif from PrtS. The  PrtH    +    and  PrtH    +    WANS  genes expression levels were measured by RT-qPCR in the corresponding mutants and compared to that of  prtS  gene in the strain LMD-9. The expression levels of both fused  prtH  CEPs genes, regardless of the anchoring motif, reached up-to more than 76% of the wild-type  prtS  expression level. CEPs were sought and identified on the cell surface of LMD-9 wild-type strain, PrtH +  and PrtH + WANS mutants using shaving technique followed by peptide identification with tandem mass spectrometry, demonstrating that the heterologous secretion and anchoring of a protein of more than 200 kDa was efficient. The anchoring to the cell-wall seems to be more efficient when the LPXTG motif of PrtS was used instead of the S-layer motif of PrtH.    Conclusions  We demonstrated  S. thermophilus  LMD-9 could heterologously secrete a high molecular weight protein and probably covalently anchor it to the cell-wall.

The naturally competent strain Streptococcus thermophilus LMD-9 as a new tool to anchor heterologous proteins on the cell surface