title: Display of a β-mannanase and a chitosanase on the cell surface of Lactobacillus plantarum towards the development of whole-cell biocatalysts.
abstract: BACKGROUND
  Lactobacillus plantarum is considered as a potential cell factory because of its GRAS (generally recognized as safe) status and long history of use in food applications. Its possible applications include in situ delivery of proteins to a host, based on its ability to persist at mucosal surfaces of the human intestine, and the production of food-related enzymes. By displaying different enzymes on the surface of L. plantarum cells these could be used as whole-cell biocatalysts for the production of oligosaccharides. In this present study, we aimed to express and display a mannanase and a chitosanase on the cell surface of L. plantarum.
  
  
  RESULTS
  ManB, a mannanase from Bacillus licheniformis DSM13, and CsnA, a chitosanase from Bacillus subtilis ATCC 23857 were fused to different anchoring motifs of L. plantarum for covalent attachment to the cell surface, either via an N-terminal lipoprotein anchor (Lp_1261) or a C-terminal cell wall anchor (Lp_2578), and the resulting fusion proteins were expressed in L. plantarum WCFS1. The localization of the recombinant proteins on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest mannanase and chitosanase activities obtained for displaying L. plantarum cells were 890 U and 1360 U g dry cell weight, respectively. In reactions with chitosan and galactomannans, L. plantarum CsnA- and ManB-displaying cells produced chito- and manno-oligosaccharides, respectively, as analyzed by high performance anion exchange chromatography (HPAEC) and mass spectrometry (MS). Surface-displayed ManB is able to break down galactomannan (LBG) into smaller manno-oligosaccharides, which can support growth of L. plantarum.
  
  
  CONCLUSIONS
  This study shows that mannanolytic and chitinolytic enzymes can be anchored to the cell surface of L. plantarum in active forms. L. plantarum chitosanase- and mannanase-displaying cells should be of interest for the production of potentially 'prebiotic' oligosaccharides. This approach, with the enzyme of interest being displayed on the cell surface of a food-grade organism, may also be applied in production processes relevant for food industry.
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18, A-1190, Vienna, Austria.|BioToP the International Doctoral Programme on Biomolecular Technology of Proteins, Muthgasse 18, A-1190, Vienna, Austria.|Department of Biotechnology, DUT-Danang University of Technology, Nguyen Luong Bang, 54, Danang, Vietnam.
authors.forename: Hoang-Minh
authors.initials: HM
authors.lastname: Nguyen
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, 1432, Ås, Norway.
authors.forename: Geir
authors.initials: G
authors.lastname: Mathiesen
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18, A-1190, Vienna, Austria.
authors.forename: Elena Maria
authors.initials: EM
authors.lastname: Stelzer
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18, A-1190, Vienna, Austria.
authors.forename: Mai Lan
authors.initials: ML
authors.lastname: Pham
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, 1432, Ås, Norway.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Kuczkowska
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, 1432, Ås, Norway.
authors.forename: Alasdair
authors.initials: A
authors.lastname: Mackenzie
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, 1432, Ås, Norway.
authors.forename: Jane W
authors.initials: JW
authors.lastname: Agger
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, 1432, Ås, Norway.
authors.forename: Vincent G H
authors.initials: VG
authors.lastname: Eijsink
authors.affiliation: Molecular Biotechnology Laboratory, School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
authors.forename: Montarop
authors.initials: M
authors.lastname: Yamabhai
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18, A-1190, Vienna, Austria.|BioToP the International Doctoral Programme on Biomolecular Technology of Proteins, Muthgasse 18, A-1190, Vienna, Austria.
authors.forename: Clemens K
authors.initials: CK
authors.lastname: Peterbauer
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18, A-1190, Vienna, Austria.|BioToP the International Doctoral Programme on Biomolecular Technology of Proteins, Muthgasse 18, A-1190, 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, Muthgasse 18, A-1190, Vienna, Austria. thu-ha.nguyen@boku.ac.at.
authors.forename: Thu-Ha
authors.initials: TH
authors.lastname: Nguyen
mesh_terms: D055162:Biocatalysis; D061505:Cell Surface Display Techniques; D003001:Cloning, Molecular; D019649:Food Industry / Q000379:methods; D005690:Galactose / Q000031:analogs & derivatives; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D048191:Lactiplantibacillus plantarum / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D008074:Lipoproteins / Q000378:metabolism; D008351:Mannans / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D056692:Prebiotics; D011994:Recombinant Proteins / Q000378:metabolism; D044902:beta-Mannosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D008074:Lipoproteins; D008351:Mannans; D009844:Oligosaccharides; D056692:Prebiotics; D011994:Recombinant Proteins; C012990:galactomannan; D006026:Glycoside Hydrolases; C019868:chitosanase; D044902:beta-Mannosidase; D005690:Galactose
keywords: Cell wall anchor; Cell-surface display; Chitosanase; Lactobacillus plantarum; Lipoprotein anchor; Mannanase; Whole-cell biocatalyst