Identification and characterization of a moonlighting protein-enolase for surface display in Streptococcus...

Streptococcus thermophilus is an important food starter and receiving more attention to serve as cell factories for production of high-valued metabolites. However, the low yields of intracellular or extracellular expression of biotechnological and biomedical proteins limit its practical applications.

Here, an enolase EnoM was identified from S. thermophilus CGMCC7.179 with about 94% identities to the surface-located enolases from other Streptococcus spp. strains. The EnoM was used as an anchor to achieve surface display in S. thermophilus using GFP as a reporter. After respectively mixing the GFP-EnoM fusion protein or GFP with S. thermophilus cells in vitro, the relative fluorescence units (RFU) of the S. thermophilus cells with GFP-EnoM was 80-folds higher than that with purified GFP. The sharp decrease in the RFU of sodium dodecyl sulfate (SDS) pretreated cells compared to those of non-pretreated cells demonstrated that the membrane proteins were the binding ligand of EnoM. Furthermore, an engineered β-galactosidase (β-Gal) was also successfully displayed on the cell surface of S. thermophilus CGMCC7.179 and the relative activity of the immobilized β-Gal remained up to 64% after reused 8 times. Finally, we also demonstrated that EnoM could be used as an anchor for surface display in L. casei, L. bulgaricus, L. lactis and Leuconostoc lactis.

To our knowledge, EnoM from S. thermophilus was firstly identified as an anchor and successfully achieved surface display in LAB. The EnoM-based surface display system provided a novel strategy for the enzyme immobilization.