Engineering Signal Peptides for Enhanced Protein Secretion from Lactococcus lactis

Abstract

bLactococcus 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 ␣-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of ␣-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 ␣-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.

A review on Lactococcus lactis: from food to factory

“…In L. lactis , there is only one majorly secreted protein which is Usp45, whose function is still unknown [19]. Nevertheless, the native lactococcal Usp45 SP is the most successful SP used thus far for secretion in L. lactis, and was recently engineered through a series of mutations to further increase its secretion efficiency (SE) by 51% [24]. More recently, we have isolated a novel signal peptide, SPK1 from Pediococcus pentasaceus , with the ability to secrete heterologous proteins with efficiencies comparable to Usp45 in L. lactis [25].…”

pMPES: A Modular Peptide Expression System for the Delivery of Antimicrobial Peptides to the Site of Gastrointestinal Infections Using Probiotics

“…The use of alternative signal peptides is a common approach for improving heterologous secretion [22,48,49,50]. We thus compared the activities of MccL, McnN and EntA constructs employing their naturally-encoded signal peptides versus the MccV signal peptide.…”

Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans

“…However, the use of the SP usp45 allowed us to achieve levels of DsrI similar to the parent strain prior to any plasmid or growth optimizations. While we did not confirm that the N-terminus of the processed DsrI was identical to that produced by the parent strain, the SP usp45 ∷DsrI fusion was designed to release a mature protein with the same sequence as that from the parent strain (Côté and Skory 2012;Ng and Sarkar 2013;van Asseldonk et al 1993).…”