Secretion of Bacillus Hydrolytic Enzymes using Food-grade Expression

System

Montarop Yamabhai1*, Peenida Namvijitra1, Suttipong Sak-Ubol1, Dietmar Haltrich2

1School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand

2Laboratory of Food Biotechnology, BOKU University of Natural Resources and Life Sciences, Vienna, Austria

Beta-mannanase, ManB

Chitosanase, Csn

Food-grade expression systems

• Bacillus subtilis

• E. coli K12

• Lactic acid bacteria (LAB)

– NICE system : Lactococcus lactis

– pSIP-based system : Lactobacillus spp.

• pSIP409, pSIP609

• Lactobacillus plantarum

Lactobacillus plantarum Expression System

• Food-grade bacteria • Generally recognized as safe

status (GRAS) • Probiotics • No endotoxins • No inclusion bodies • Simple cultivation, non-aerated

fermentation • Complex nutritional

requirements • Acidifying culture, required pH-

control

pSIP-based expression system

Diep, D.B., Mathiesen, G., Eijsink, V.G., and Nes, I.F. (2009). Use of lactobacilli and their pheromone-based regulatory mechanism in gene expression and drug delivery. Curr Pharm Biotechnol 10, 62-73.

• quorum-sensing mechanisms to induce gene expression, using bacteriocins (Sakacin A and Sakacin P).

• Suitable for Lactobacillus spp.

• Promoter PsapA in the pSIP300 series and PsppA or PsppQ (ProfX) in the pSIP400 series

• All components are located in one plasmid with modular design

Using Alanine Racemase (alr) for Food-grade expression

D-alanine is a component of the peptidoglycan structure of bacterial cell wall alr

Chitosanse (Csn)

Chitooligosaccharides (CHOS)

β-Mannanase (ManB)

Glucomannan, Galactomannan

Mannooligosaccharides (MOS)

pSIP-based Expression Vectors

pSIP409

Mature Enzyme Beta-Mannanase Chitosanase

OmpA Native Signal P

pSIP609

OmpA Native Signal P

Mature Enzyme Beta-Mannanase Chitosanase

ery: erythromycin-resistant gene alr: alanine racemase gene

Lactobacillus plantarum WCFS1 Lactobacillus plantarum TLG02 (Δalr, D - alanine auxotroph) Nguyen et al, (2011). J Agric Food Chem 59, 5617-5624.

Csn

Csn

Native signal peptide

OmpA signal peptide

pSIP609 (alr) Food-grade expression

β-Mannanase (ManB)

ManB

ManB

Native signal peptide

OmpA signal peptide

Rep1

Rep2

Rep3

E.coli 27700* 24500 53.1 %

* 14200 (broth) + 13500 (periplasmic space)

Conclusions

• Two bacillus hydrolytic enzymes (Csn and ManB) were successfully expressed in L. plantarum food-grade expression system, using pSIP-based vector

• Both E. coli OmpA and bacillus native signal peptides can direct the secretion of both enzymes into culture broth; however, native signal peptide is more efficient

Kob-khun-ka Thank you

Professor Vincent Eijsink (The Norwegian University of Life Sciences)

NRU