Abstract
BackgroundLactic 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.ResultsTo 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.ConclusionsOverall, 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.
Highlights
Lactic acid bacteria are a family of “generally regarded as safe” organisms traditionally used for food fermentation
Besides being the currently most utilized secretion signal peptide for L. lactis, USP45 SP has been shown to be more efficient over other natural signal peptides (SP310 [24]) or mutated libraries (USP45MT11 [14], SP310mut2 [17])
From the BlastP search, three propeptide sequences, which were most representative of the results, were identified (Fig. 1a)
Summary
Lactic acid bacteria are a family of “generally regarded as safe” organisms traditionally used for food fermentation. Studies of LAB and host interactions have associated LAB directly with cellular activities of the gut, such as pathogen control, immunostimulation and maintaining a healthy microflora [3, 4] Combined with their traditional roles in food fermentation, beneficial gut properties, and resistance to harsh gut conditions, additional advantages such as LAB’s ability to secrete recombinant proteins while possessing fewer proteases, compared to traditional workhorses, such as Escherichia coli and Bacillus subtilis, have made them attractive targets for recombinant cell factories and live vectors for delivery of therapeutic molecules in the gut [5]. A recent promising development of oral therapy for celiac disease, using food grade prolyl endopeptidase (PEP) to break down contaminant gluten, have sparked interest into exploring non-dietary alternatives using mucosal enzyme delivery vectors, such as LAB. Demonstration of secreted and functional Fm PEP from LAB would be a significant step for the prospective treatment of celiac disease
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