title: Secretory production of a beta-mannanase and a chitosanase using a Lactobacillus plantarum expression system.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  ManB 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.
authors.affiliation: Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand.|Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Suttipong
authors.initials: S
authors.lastname: Sak-Ubol
authors.affiliation: Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
authors.forename: Peenida
authors.initials: P
authors.lastname: Namvijitr
authors.affiliation: Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
authors.forename: Phornsiri
authors.initials: P
authors.lastname: Pechsrichuang
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Haltrich
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Thu-Ha
authors.initials: TH
authors.lastname: Nguyen
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), Ås, Norway.
authors.forename: Geir
authors.initials: G
authors.lastname: Mathiesen
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), Ås, Norway.
authors.forename: Vincent G H
authors.initials: VG
authors.lastname: Eijsink
authors.affiliation: Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand. Montarop@sut.ac.th.
authors.forename: Montarop
authors.identifier: http://orcid.org/0000-0003-2674-2419
authors.initials: M
authors.lastname: Yamabhai
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D048191:Lactiplantibacillus plantarum / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D044902:beta-Mannosidase / Q000235:genetics* / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C078167:OMPA outer membrane proteins; D006026:Glycoside Hydrolases; C019868:chitosanase; D044902:beta-Mannosidase
keywords: Alanine racemase; Chitosanase; Food-grade, Bacillus; L. plantarum; OmpA; Secretion; Signal peptide; pSIP; β-Mannanase