Secretory production of a beta-mannanase and a chitosanase using a Lactobacillus plantarum expression system.

Suttipong Sak-Ubol,Vincent G H Eijsink,Geir Mathiesen,Thu-Ha Nguyen,Peenida Namvijitr,Phornsiri Pechsrichuang,Montarop Yamabhai,Dietmar Haltrich

doi:10.1186/s12934-016-0481-z

Abstract

BackgroundHeterologous production of hydrolytic enzymes is important for green and white biotechnology since these enzymes serve as efficient biocatalysts for the conversion of a wide variety of raw materials into value-added products. Lactic acid bacteria are interesting cell factories for the expression of hydrolytic enzymes as many of them are generally recognized as safe and require only a simple cultivation process. We are studying a potentially food-grade expression system for secretion of hydrolytic enzymes into the culture medium, since this enables easy harvesting and purification, while allowing direct use of the enzymes in food applications.ResultsWe studied overexpression of a chitosanase (CsnA) and a β-mannanase (ManB), from Bacillus licheniformis and Bacillus subtilis, respectively, in Lactobacillus plantarum, using the pSIP system for inducible expression. The enzymes were over-expressed in three forms: without a signal peptide, with their natural signal peptide and with the well-known OmpA signal peptide from Escherichia coli. The total production levels and secretion efficiencies of CsnA and ManB were highest when using the native signal peptides, and both were reduced considerably when using the OmpA signal. At 20 h after induction with 12.5 ng/mL of inducing peptide in MRS media containing 20 g/L glucose, the yields and secretion efficiencies of the proteins with their native signal peptides were 50 kU/L and 84 % for ManB, and 79 kU/L and 56 % for CsnA, respectively. In addition, to avoid using antibiotics, the erythromycin resistance gene was replaced on the expression plasmid with the alanine racemase (alr) gene, which led to comparable levels of protein production and secretion efficiency in a suitable, alr-deficient L. plantarum host.ConclusionsManB and CsnA were efficiently produced and secreted in L. plantarum using pSIP-based expression vectors containing either an erythromycin resistance or the alr gene as selection marker.

Highlights

Heterologous production of hydrolytic enzymes is important for green and white biotechnology since these enzymes serve as efficient biocatalysts for the conversion of a wide variety of raw materials into value-added products
Each gene was cloned in three forms: with no signal peptide (BlManB_noSP and BsCsnA_noSP), with their native signal peptides (BlManB_nt and BsCsnA_nt) or with the E. coli OmpA signal peptide (BlManB_OmpA and BsCsnA_OmpA)
Enzyme activities were determined in both culture supernatants and cell lysates to calculate volumetric activities at different time points during the cultivations (Fig. 2; Table 1). Both Bacillus enzymes were secreted when using either of the two signal peptides, yet using the native signal peptides resulted in both the highest total production levels and the highest secretion efficiencies. While these data show that the OmpA signal peptide from Gram-negative E. coli does function in Lactobacillus, they indicate that signal peptides from Gram-positive bacilli work better

Summary

Introduction

Heterologous production of hydrolytic enzymes is important for green and white biotechnology since these enzymes serve as efficient biocatalysts for the conversion of a wide variety of raw materials into value-added products. In the present study we selected two extracellular, hydrolytic enzymes from Bacillus, a β-mannanase from Bacillus licheniformis (BlManB) and a chitosanase from Bacillus subtilis (BsCsnA), to study secretory production in L. plantarum. Both enzymes are of interest for biotechnological applications, namely the conversion of hemicelluloses (mannans) and chitosan into mannooligosaccharides (MOS) [9] and chito-oligosaccharides (CHOS) [10], respectively. For comparative purposes, these enzymes were overproduced in three forms: with no signal peptide, with their native (Bacillus) signal peptide, and with a signal peptide derived from the Escherichia coli OmpA protein. The engineered production strains were evaluated in terms of enzyme yields and secretion efficiencies

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Conclusion