Login | Register

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis


Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

Wieczorek, Andrew S and Martin, Vincent JJ (2010) Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis. Microbial Cell Factories, 9 (1). p. 69. ISSN 1475-2859

[thumbnail of martin_microbialcellfactories2010.pdf]
Text (application/pdf)
martin_microbialcellfactories2010.pdf - Published Version

Official URL: http://dx.doi.org/10.1186/1475-2859-9-69


The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes.

Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell.

We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals.

Divisions:Concordia University > Faculty of Arts and Science > Biology
Item Type:Article
Authors:Wieczorek, Andrew S and Martin, Vincent JJ
Journal or Publication:Microbial Cell Factories
Digital Object Identifier (DOI):10.1186/1475-2859-9-69
ID Code:6994
Deposited By: Danielle Dennie
Deposited On:17 Dec 2010 15:52
Last Modified:18 Jan 2018 17:29


1.Lowell GH, Ballou WR, Smith LF, Wirtz RA, Zollinger WD, Hockmeyer WT: Proteosome-lipopeptide vaccines: enhancement of immunogenicity for malaria CS peptides. Science 1988, 240:800-802.

2.Lowell GH, Smith LF, Seid RC, Zollinger WD: Peptides bound to proteosomes via hydrophobic feet become highly immunogenic without adjuvants. J Exp Med 1988, 167:658-663.

3.Bayer EA, Belaich JP, Shoham Y, Lamed R: The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides. Annu Rev Microbiol 2004 , 58:521-554.

4.Conrado RJ, Varner JD, DeLisa MP: Engineering the spatial organization of metabolic enzymes: mimicking nature's synergy. Curr Opin Biotechnol 2008 , 19:492-499.

5.Dueber JE, Wu GC, Malmirchegini GR, Moon TS, Petzold CJ, Ullal AV, Prather KL, Keasling JD: Synthetic protein scaffolds provide modular control over metabolic flux. Nat Biotechnol 2009 , 27:753-759. PubMed

6.Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS: Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 2002 , 66:506-577.

7.Schwarz WH: The cellulosome and cellulose degradation by anaerobic bacteria. Appl Microbiol Biotechnol 2001 , 56:634-649.

8.Kruus K, Lua AC, Demain AL, Wu JH: The anchorage function of CipA (CelL), a scaffolding protein of the Clostridium thermocellum cellulosome. Proc Natl Acad Sci USA 1995 , 92:9254-9258.

9.Leibovitz E, Beguin P: A new type of cohesin domain that specifically binds the dockerin domain of the Clostridium thermocellum cellulosome-integrating protein CipA. J Bacteriol 1996 , 178:3077-3084.

10.Lemaire M, Ohayon H, Gounon P, Fujino T, Beguin P: OlpB, a new outer layer protein of Clostridium thermocellum, and binding of its S-layer-like domains to components of the cell envelope. J Bacteriol 1995 , 177:2451-2459.

11.Kosugi A, Amano Y, Murashima K, Doi RH: Hydrophilic domains of scaffolding protein CbpA promote glycosyl hydrolase activity and localization of cellulosomes to the cell surface of Clostridium cellulovorans. J Bacteriol 2004 , 186:6351-6359.

12.Garcia-Campayo V, Beguin P: Synergism between the cellulosome-integrating protein CipA and endoglucanase CelD of Clostridium thermocellum. J Biotechnol 1997 , 57:39-47.

13.Zverlov VV, Klupp M, Krauss J, Schwarz WH: Mutations in the scaffoldin gene, cipA, of Clostridium thermocellum with impaired cellulosome formation and cellulose hydrolysis: insertions of a new transposable element, IS1447, and implications for cellulase synergism on crystalline cellulose. J Bacteriol 2008 , 190:4321-4327.

14.Lynd LR, van Zyl WH, McBride JE, Laser M: Consolidated bioprocessing of cellulosic biomass: an update. Curr Opin Biotechnol 2005 , 16:577-583.

15.Lu Y, Zhang YH, Lynd LR: Enzyme-microbe synergy during cellulose hydrolysis by Clostridium thermocellum. Proc Natl Acad Sci USA 2006 , 103:16165-16169.

16.Miron J, Ben-Ghedalia D, Morrison M: Invited review: adhesion mechanisms of rumen cellulolytic bacteria. J Dairy Sci 2001 , 84:1294-1309.

17.Bayer EA, Kenig R, Lamed R: Adherence of Clostridium thermocellum to cellulose. J Bacteriol 1983 , 156:818-827.

18.Ng TK, Weimer TK, Zeikus JG: Cellulolytic and physiological properties of Clostridium thermocellum. Arch Microbiol 1977 , 114:1-7.

19.Fierobe HP, Bayer EA, Tardif C, Czjzek M, Mechaly A, Belaich A, Lamed R, Shoham Y, Belaich JP: Degradation of cellulose substrates by cellulosome chimeras. Substrate targeting versus proximity of enzyme components. J Biol Chem 2002 , 277:49621-49630.

20.Fierobe HP, Mechaly A, Tardif C, Belaich A, Lamed R, Shoham Y, Belaich JP, Bayer EA: Design and production of active cellulosome chimeras. Selective incorporation of dockerin-containing enzymes into defined functional complexes. J Biol Chem 2001 , 276:21257-21261.

21.Fierobe HP, Mingardon F, Mechaly A, Belaich A, Rincon MT, Pages S, Lamed R, Tardif C, Belaich JP, Bayer EA: Action of designer cellulosomes on homogeneous versus complex substrates: controlled incorporation of three distinct enzymes into a defined trifunctional scaffoldin. J Biol Chem 2005 , 280:16325-16334.

22.Mingardon F, Chanal A, Tardif C, Bayer EA, Fierobe HP: Exploration of new geometries in cellulosome-like chimeras. Appl Environ Microbiol 2007 , 73:7138-7149.

23.Murashima K, Kosugi A, Doi RH: Synergistic effects on crystalline cellulose degradation between cellulosomal cellulases from Clostridium cellulovorans. J Bacteriol 2002 , 184:5088-5095.

24.Perret S, Casalot L, Fierobe HP, Tardif C, Sabathe F, Belaich JP, Belaich A: Production of heterologous and chimeric scaffoldins by Clostridium acetobutylicum ATCC 824. J Bacteriol 2004 , 186:253-257. PubMed Abstract | Publisher

25.Sabathe F, Soucaille P: Characterization of the CipA scaffolding protein and in vivo production of a minicellulosome in Clostridium acetobutylicum. J Bacteriol 2003 , 185:1092-1096.

26.Ito J, Kosugi A, Tanaka T, Kuroda K, Shibasaki S, Ogino C, Ueda M, Fukuda H, Doi RH, Kondo A: Regulation of the display ratio of enzymes on the Saccharomyces cerevisiae cell surface by the immunoglobulin G and cellulosomal enzyme binding domains. Appl Environ Microbiol 2009 , 75:4149-4154.

27.Tsai SL, Oh J, Singh S, Chen R, Chen W: Functional assembly of minicellulosomes on the Saccharomyces cerevisiae cell surface for cellulose hydrolysis and ethanol production. Appl Environ Microbiol 2009 , 75:6087-6093.

28.Lilly M, Fierobe HP, van Zyl WH, Volschenk H: Heterologous expression of a Clostridium minicellulosome in Saccharomyces cerevisiae. FEMS Yeast Res 2009 , 9:1236-1249.

29.Wen F, Sun J, Zhao H: Yeast surface display of trifunctional minicellulosomes for simultaneous saccharification and fermentation of cellulose to ethanol. Appl Environ Microbiol 76:1251-1260.

30.Petrov K, Urshev Z, Petrova P: L+-lactic acid production from starch by a novel amylolytic Lactococcus lactis subsp. lactis B84. Food Microbiol 2008 , 25:550-557.

31.Hernandez I, Molenaar D, Beekwilder J, Bouwmeester H, van Hylckama Vlieg JE: Expression of plant flavor genes in Lactococcus lactis. Appl Environ Microbiol 2007 , 73:1544-1552.

32.Le Loir Y, Azevedo V, Oliveira SC, Freitas DA, Miyoshi A, Bermudez-Humaran LG, Nouaille S, Ribeiro LA, Leclercq S, Gabriel JE, et al.: Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production. Microb Cell Fact 2005 , 4:2.

33.Narita J, Okano K, Kitao T, Ishida S, Sewaki T, Sung MH, Fukuda H, Kondo A: Display of alpha-amylase on the surface of Lactobacillus casei cells by use of the PgsA anchor protein, and production of lactic acid from starch. Appl Environ Microbiol 2006 , 72:269-275.

34.Zhang YH, Lynd LR: Regulation of cellulase synthesis in batch and continuous cultures of Clostridium thermocellum.
J Bacteriol 2005 , 187:99-106.

35.Dieye Y, Hoekman AJ, Clier F, Juillard V, Boot HJ, Piard JC: Ability of Lactococcus lactis to export viral capsid antigens: a crucial step for development of live vaccines.
Appl Environ Microbiol 2003 , 69:7281-7288.

36.Dieye Y, Usai S, Clier F, Gruss A, Piard JC: Design of a protein-targeting system for lactic acid bacteria. J Bacteriol 2001 , 183:4157-4166.

37.Miyoshi A, Poquet I, Azevedo V, Commissaire J, Bermudez-Humaran L, Domakova E, Le Loir Y, Oliveira SC, Gruss A, Langella P: Controlled production of stable heterologous proteins in Lactococcus lactis. Appl Environ Microbiol 2002 , 68:3141-3146.

38.Ribeiro LA, Azevedo V, Le Loir Y, Oliveira SC, Dieye Y, Piard JC, Gruss A, Langella P: Production and targeting of the Brucella abortus antigen L7/L12 in Lactococcus lactis: a first step towards food-grade live vaccines against brucellosis. Appl Environ Microbiol 2002 , 68:910-916.

39.Langella P, Le Loir Y: Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system. Braz J Med Biol Res 1999 , 32:191-198.

40.Atsumi S, Hanai T, Liao JC: Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 2008 , 451:86-89.

41.Zhang M, Eddy C, Deanda K, Finkelstein M, Picataggio S: Metabolic engineering of a pentose M\metabolism pathway in ethanologenic Zymomonas mobilis. Science 1995 , 267:240-243.

42.Wu CH, Mulchandani A, Chen W: Versatile microbial surface-display for environmental remediation and biofuels production. Trends Microbiol 2008 , 16:181-188.

43.Rittmann D, Lindner SN, Wendisch VF: Engineering of a glycerol utilization pathway for amino acid production by Corynebacterium glutamicum. Appl Environ Microbiol 2008 , 74:6216-6222.

44.Lee SK, Chou H, Ham TS, Lee TS, Keasling JD: Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels. Curr Opin Biotechnol 2008 , 19:556-563.

45.Rogers PL, Jeon YJ, Lee KJ, Lawford HG: Zymomonas mobilis for fuel ethanol and higher value products. Adv Biochem Eng Biotechnol 2007 , 108:263-288.

46.Steen EJ, Kang Y, Bokinsky G, Hu Z, Schirmer A, McClure A, Del Cardayre SB, Keasling JD: Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 463:559-562.

47.Shaw AJ, Podkaminer KK, Desai SG, Bardsley JS, Rogers SR, Thorne PG, Hogsett DA, Lynd LR: Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield. Proc Natl Acad Sci USA 2008 , 105:13769-13774.

48.de Vos WM: Gene expression systems for lactic acid bacteria. Curr Opin Microbiol 1999 , 2:289-295.

49.Bermudez-Humaran LG, Cortes-Perez NG, Le Loir Y, Alcocer-Gonzalez JM, Tamez-Guerra RS, de Oca-Luna RM, Langella P: An inducible surface presentation system improves cellular immunity against human papillomavirus type 16 E7 antigen in mice after nasal administration with recombinant lactococci. J Med Microbiol 2004 , 53:427-433.

50.Leenhouts K, Buist G, Kok J: Anchoring of proteins to lactic acid bacteria. Antonie Van Leeuwenhoek 1999 , 76:367-376.

51.Gerngross UT, Romaniec MP, Kobayashi T, Huskisson NS, Demain AL: Sequencing of a Clostridium thermocellum gene (cipA) encoding the cellulosomal SL-protein reveals an unusual degree of internal homology. Mol Microbiol 1993 , 8:325-334.

52.Lytle B, Myers C, Kruus K, Wu JH: Interactions of the CelS binding ligand with various receptor domains of the Clostridium thermocellum cellulosomal scaffolding protein, CipA. J Bacteriol 1996 , 178:1200-1203.

53.Murashima K, Kosugi A, Doi RH: Solubilization of cellulosomal cellulases by fusion with cellulose-binding domain of noncellulosomal cellulase engd from Clostridium cellulovorans. Proteins 2003 , 50:620-628.

54.Bermudez-Humaran LG, Langella P, Miyoshi A, Gruss A, Guerra RT, Montes de Oca-Luna R, Le Loir Y: Production of human papillomavirus type 16 E7 protein in Lactococcus lactis. Appl Environ Microbiol 2002 , 68:917-922.

55.Avall-Jaaskelainen S, Lindholm A, Palva A: Surface display of the receptor-binding region of the Lactobacillus brevis S-layer protein in Lactococcus lactis provides nonadhesive lactococci with the ability to adhere to intestinal epithelial cells. Appl Environ Microbiol 2003 , 69:2230-2236.

56.Cortes-Perez NG, Azevedo V, Alcocer-Gonzalez JM, Rodriguez-Padilla C, Tamez-Guerra RS, Corthier G, Gruss A, Langella P, Bermudez-Humaran LG: Cell-surface display of E7 antigen from human papillomavirus type-16 in Lactococcus lactis and in Lactobacillus plantarum using a new cell-wall anchor from lactobacilli. J Drug Target 2005 , 13:89-98.

57.Lindholm A, Smeds A, Palva A: Receptor binding domain of Escherichia coli F18 fimbrial adhesin FedF can be both efficiently secreted and surface displayed in a functional form in Lactococcus lactis. Appl Environ Microbiol 2004 , 70:2061-2071.

58.Piard JC, Hautefort I, Fischetti VA, Ehrlich SD, Fons M, Gruss A: Cell wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria. J Bacteriol 1997 , 179:3068-3072.

59.Raha AR, Varma NR, Yusoff K, Ross E, Foo HL: Cell surface display system for Lactococcus lactis: a novel development for oral vaccine. Appl Microbiol Biotechnol 2005 , 68:75-81.

60.Ramasamy R, Yasawardena S, Zomer A, Venema G, Kok J, Leenhouts K: Immunogenicity of a malaria parasite antigen displayed by Lactococcus lactis in oral immunisations.
Vaccine 2006 , 24:3900-3908.

61.Yang Z, Liu Q, Wang Q, Zhang Y: Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium Vibrio anguillarum. Appl Environ Microbiol 2008 , 74:4359-4365.

62.Terzaghi BE, Sandine WE: Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol 1975 , 29:807-813.

63.Sambrook J, Russell DW: Molecular cloning: a laboratory manual. 3rd edition. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press; 2001.

64.Wang WK, Wu JH: Structural features of the Clostridium thermocellum cellulase SS gene. Appl Biochem Biotechnol 1993 , 39-40:149-158.

65.Holo H, Nes IF: High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl Environ Microbiol 1989 , 55:3119-3123.

66.Sorvig E, Gronqvist S, Naterstad K, Mathiesen G, Eijsink VG, Axelsson L: Construction of vectors for inducible gene expression in Lactobacillus sakei and L plantarum. FEMS Microbiol Lett 2003 , 229:119-126.

67.Steidler L, Viaene J, Fiers W, Remaut E: Functional display of a heterologous protein on the surface of Lactococcus lactis by means of the cell wall anchor of Staphylococcus aureus protein A. Appl Environ Microbiol 1998 , 64:342-345.

68.Axelsson L, Lindstad G, Naterstad K: Development of an inducible gene expression system for Lactobacillus sakei. Lett Appl Microbiol 2003 , 37:115-120.

69.Yanisch-Perron C, Vieira J, Messing J: Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 1985 , 33:103-119
All items in Spectrum are protected by copyright, with all rights reserved. The use of items is governed by Spectrum's terms of access.

Repository Staff Only: item control page

Downloads per month over past year

Research related to the current document (at the CORE website)
- Research related to the current document (at the CORE website)
Back to top Back to top