title: Engineering signal peptides for enhanced protein secretion from Lactococcus lactis.
abstract: Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.
authors.affiliation: Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Daphne T W
authors.initials: DT
authors.lastname: Ng
authors.forename: Casim A
authors.initials: CA
authors.lastname: Sarkar
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001709:Biotechnology / Q000379:methods; D004252:DNA Mutational Analysis; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D060847:Metabolic Engineering; D008836:Micrococcal Nuclease / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics; D013678:Technology, Pharmaceutical / Q000379:methods; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism
chemical_list: D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D008836:Micrococcal Nuclease; D000516:alpha-Amylases