Abstract
Lactic acid bacteria (LAB) have attracted increasing interest recently as cell factories for the production of proteins as well as a carrier of proteins that are of interest for food and therapeutic applications. In this present study, we exploit a lactobacillal food-grade expression system derived from the pSIP expression vectors using the alr (alanine racemase) gene as the selection marker for the expression and cell-surface display of a chitosanase in Lactobacillus plantarum using two truncated forms of a LP × TG anchor. CsnA, a chitosanase from Bacillus subtilis 168 (ATCC23857), was fused to two different truncated forms (short-S and long-L anchors) of an LP × TG anchor derived from Lp_1229, a key-protein for mannose-specific adhesion in L. plantarum WCFS1. The expression and cell-surface display efficiency driven by the food-grade alr-based system were compared with those obtained from the erm-based pSIP system in terms of enzyme activities and their localisation on L. plantarum cells. The localization of the protein on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest enzymatic activity of CsnA-displaying cells was obtained from the strain carrying the alr-based expression plasmid with short cell wall anchor S. However, the attachment of chitosanase on L. plantarum cells via the long anchor L was shown to be more stable compared with the short anchor after several repeated reaction cycles. CsnA displayed on L. plantarum cells is catalytically active and can convert chitosan into chito-oligosaccharides, of which chitobiose and chitotriose are the main products.
Highlights
Chitin, which can be found in the outer exoskeleton of arthropods, such as crabs, lobster, shrimp, and in the fungal cell wall, is the second most abundant biopolymer after lignocellulose in nature [1]
We reported cell-surface display of a chitosanase via a lipoprotein anchor (Lp_1261) and an LP × TG motif-containing a cell wall anchor from a cell surface adherence protein (Lp_2578) of L. plantarum WCFS1 [22], using the original erm-based pSIP expression system [23,24]
Four new expression vectors were constructed to display a chitosanase from Bacillus subtilis 168 on the surface of L. plantarum (Figure 1A–C) using both erm-based and alr-based expression systems in lactobacilli
Summary
Chitin, which can be found in the outer exoskeleton of arthropods, such as crabs, lobster, shrimp, and in the fungal cell wall, is the second most abundant biopolymer after lignocellulose in nature [1]. The chitosanase from B. subtilis 168, which is encoded by csnA gene, was demonstrated to be efficiently produced and secreted in Lactobacillus plantarum using the inducible promoter-based pSIP expression vectors containing either an antibiotic resistance or the alr gene as selection markers [11]. We reported cell-surface display of a chitosanase via a lipoprotein anchor (Lp_1261) and an LP × TG motif-containing a cell wall anchor from a cell surface adherence protein (Lp_2578) of L. plantarum WCFS1 [22], using the original erm-based pSIP expression system [23,24]. The expression and cell-surface display efficiency driven by the food-grade system were compared with those obtained from the erm-based pSIP system in term of enzyme activities and their localisation on L. plantarum cells
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