A nine-residue synthetic propeptide enhances secretion efficiency of heterologous proteins in Lactococcus lactis

Lactococcus lactis, a gram-positive organism widely used in the food industry, is a potential candidate for the secretion of biologically useful proteins. We examined the secretion efficiency and capacity oft. lactis by using the Staphylococcus aureus nuclease (Nuc) as a heterologous model protein. When expressed in L. lactis from an efficient lactococcal promoter and its native signal peptide, only similar to 60% of total Nuc was present in a secreted form at similar to 5 mg per liter. The remaining 40% was found in a cell-associated precursor form. The secretion efficiency aas reduced further to similar to 30% by the deletion of 17 residues of the Nuc native propeptide (resulting in NucT). We identified a modification which improved secretion efficiency of both native Nuc and NucT. A 9-residue synthetic propeptide, LEISSTCDA, which adds two negative charges at the +2 and +8 positions, was fused immediately after the signal peptide cleavage site. In the case of Nuc, secretion efficiency was increased to similar to 80% by LEISSTCDA insertion without altering the signal peptide cleavage site, and the yield was increased two-to fourfold (up to similar to 20 mg per liter). The improvement of NucT secretion efficiency was even more marked and rose from 30 to 90%. Similarly, the secretion efficiency of a third protein, the cp-amylase of Bacillus stearothermophilus, was also improved by LEISSTCDA. These data indicate that the LEISSTCDA synthetic propeptide improves secretion of different heterologous proteins in L. lactis.