Engineering Streptococcus thermophilus for heterologous gene expression of cell envelope proteases from lactic acid bacteria

Abstract Background Cell envelope proteinases have long played a pivotal role in dairy science. However, as demand for alternative food sources grows, their application in plant-based food matrices is scarcely investigated. A deeper physiological and technological understanding of these enzymes requires efficient and effective tools tailored to this emerging sector. To date, plasmid-based recombination in Lactococcus spp. remains the most widely used and effective method. Results In this study, we engineered S. thermophilus LMD-9 as a host for heterologous expression of protease from Lc. cremoris . S. thermophilus LMD-9 offers several advantages, including its GRAS status, efficient chromosomal gene integration via natural competence, and native machinery for functional protease production, making it a highly versatile host. Previous attempts to employ this strain yielded inactive protease due to unresolved bottlenecks; here, we characterize and overcome those challenges to establish LMD-9 as a robust system for protease expression. Exploration of potential bottlenecks highlighted the availability of intrinsic peptidyl-prolyl cis / trans isomerase (PPIase; prtM / prsA ) as a key factor influencing successful enzyme expression. Three recombinant strains with genotypes: LMD-9 Δ prtS :: ermR , LMD-9 Δ prtS :: ermR —Ω prtP , and LMD-9 Δ prtS :: ermR —Ω ( prtM-prtP ), were generated to test the role of prtP -associated PPIase. Results demonstrated that inclusion of PrtP-specific PPIase from L. cremoris markedly enhances protease activity. In its absence, although partially compensated by the pleiotropic PPIase in S. thermophilus , we observed slower growth and reduced proteolytic activity. Conclusions These findings establish S. thermophilus LMD-9 as a robust chassis and alternative host for heterologous expression of cell envelope proteinases. To our knowledge, this is the first work to heterologously express active CEP enzyme in this host, and it highlights the role of PPIase availability as a key determinant of successful enzyme expression. This then provides a suitable and robust host for studying the application of CEPs in both dairy and emerging plant-based food applications.