Heterologous expression of a recombinant lactobacillal β-galactosidase in Lactobacillus plantarum: effect of different parameters on the sakacin P-based expression system.

Tien-Thanh Nguyen,Clemens K Peterbauer,Barbara Geiger,Dietmar Haltrich,Thu-Ha Nguyen,Vincent Eijsink,Hoang-Minh Nguyen,Geir Mathiesen

doi:10.1186/s12934-015-0214-8

Abstract

BackgroundTwo overlapping genes lacL and lacM (lacLM) encoding for heterodimeric β-galactosidase from Lactobacillus reuteri were previously cloned and over-expressed in the food-grade host strain Lactobacillus plantarum WCFS1, using the inducible lactobacillal pSIP expression system. In this study, we analyzed different factors that affect the production of recombinant L. reuteri β-galactosidase.ResultsVarious factors related to the cultivation, i.e. culture pH, growth temperature, glucose concentration, as well as the induction conditions, including cell concentration at induction point and inducer concentration, were tested. Under optimal fermentation conditions, the maximum β-galactosidase levels obtained were 130 U/mg protein and 35–40 U/ml of fermentation broth corresponding to the formation of approximately 200 mg of recombinant protein per litre of fermentation medium. As calculated from the specific activity of the purified enzyme (190 U/mg), β-galactosidase yield amounted to roughly 70% of the total soluble intracellular protein of the host organism. It was observed that pH and substrate (glucose) concentration are the most prominent factors affecting the production of recombinant β-galactosidase.ConclusionsThe over-expression of recombinant L. reuteri β-galactosidase in a food-grade host strain was optimized, which is of interest for applications of this enzyme in the food industry. The results provide more detailed insight into these lactobacillal expression systems and confirm the potential of the pSIP system for efficient, tightly controlled expression of enzymes and proteins in lactobacilli.

Highlights

Two overlapping genes lacL and lacM encoding for heterodimeric β-galactosidase from Lactobacillus reuteri were previously cloned and over-expressed in the food-grade host strain Lactobacillus plantarum WCFS1, using the inducible lactobacillal pSIP expression system
Some lactic acid bacteria are known as producers of processing enzymes, antimicrobial peptides, or metabolites that contribute to flavor, conservation or texture of various foods
One of the most widely used gene expression systems derived from Lactic acid bacteria (LAB) is the NIsin-Controlled gene Expression system (NICE), which is based on the autoregulatory properties and the genes involved in the synthesis of nisin, an antimicrobial peptide produced by certain strains of Lactococcus lactis [9]

Summary

Introduction

Two overlapping genes lacL and lacM (lacLM) encoding for heterodimeric β-galactosidase from Lactobacillus reuteri were previously cloned and over-expressed in the food-grade host strain Lactobacillus plantarum WCFS1, using the inducible lactobacillal pSIP expression system. The so-called pSIP system [12], was constructed for Lactobacillus spp. based on the promoter and regulatory genes involved in the production of the class-II bacteriocins sakacin A [13] and sakacin P [14,15]. In the pSIP systems, expression of the gene of interest is under control of a strong, inducible bacteriocin promoter, and gene expression is induced by external addition of the peptide pheromone An advantage of these systems is that they are strictly regulated and lead to high production of the target protein. The applicability of these sakacin-based expression systems was shown for the over-production of enzymes such as β-glucuronidase and aminopeptidase in several Lactobacillus hosts [7,12]

Methods

Results

Conclusion