Microbial Cell Factories (Dec 2017)
A propeptide toolbox for secretion optimization of Flavobacterium meningosepticum endopeptidase in Lactococcus lactis
Abstract
Abstract Background Lactic acid bacteria are a family of “generally regarded as safe” organisms traditionally used for food fermentation. In recent years, they have started to emerge as potential chassis for heterologous protein production. And more recently, due to their beneficial properties in the gut, they have been examined as potential candidates for mucosal delivery vectors, especially for acid-sensitive enzymes. One such application would be the delivery of gluten-digesting endopeptidases for the treatment of celiac disease. To facilitate these applications, an efficient recombinant protein expression toolbox is required, especially for recombinant protein secretion. While current tools for enhancing protein secretion consist mainly of signal peptides, secretion propeptides have also been observed to play a crucial role for protein secretion and improved yields. Results To expand the propeptide library for secretion optimization, we have mined and characterized three naturally occurring propeptides from the sequenced genomes of 109 Lactococcus species. These newly-mined propeptides were introduced after the N-terminal USP45 secretion signal to characterize and compare their effects on the secretion of Escherichia coli thioredoxin (TRX) and Flavobacterium meningosepticum prolyl endopeptidase (Fm PEP) in Lactococcus lactis NZ9000. All three propeptides, along with the positive control LEISSTCDA, improved volumetric secretion yields by 1.4–2.3-folds. However, enhancement of secretion yield is dependent on protein of interest. For TRX, the optimal combination of USP45 signal peptide and LEISSTCDA produced a 2.3-fold increase in secretion yields. Whilst for Fm PEP, propeptide 1 with USP45 signal peptide improved volumetric secretion yields by 2.2-fold compared to a 1.4-fold increase by LEISSTCDA. Similar trends in Fm PEP activity and protein yield also demonstrated minimal effect of the negative charged propeptides on PEP activity and thus folding. Conclusions Overall, we have characterized three new propeptides for use in L. lactis secretion optimization. From success of these propeptides for improvement of secretion yields, we anticipate this collection to be valuable to heterologous protein secretion optimisation in lactic acid bacteria. We have also demonstrated for the first time, secretion of Fm PEP in L. lactis for potential use as a therapy agent in celiac disease.
Keywords
