Abstract
Novel enzymes that are stable in diverse conditions are intensively sought because they offer major potential advantages in industrial biotechnology, and microorganisms in extreme environments are key sources of such enzymes. However, most potentially valuable enzymes are currently inaccessible due to the pure culturing problem of microorganisms. Novel metagenomic and metaproteomic techniques that circumvent the need for pure cultures have theoretically provided possibilities to identify all genes and all proteins in microbial communities, but these techniques have not been widely used to directly identify specific enzymes because they generate vast amounts of extraneous data.
In a first step towards developing a metaproteomic approach to pinpoint targeted extracellular hydrolytic enzymes of choice in microbial communities, we have generated and analyzed the necessary conditions for such an approach by the use of a methanogenic microbial community maintained on a chemically defined medium. The results show that a metabolic steady state of the microbial community could be reached, at which the expression of the targeted hydrolytic enzymes were suppressed, and that upon enzyme induction a distinct increase in the targeted enzyme expression was obtained. Furthermore, no cross talk in expression was detected between the two focal types of enzyme activities under their respective inductive conditions. Thus, the described approach should be useful to generate ideal samples, collected before and after selective induction, in controlled microbial communities to clearly discriminate between constituently expressed proteins and extracellular hydrolytic enzymes that are specifically induced, thereby reducing the analysis to only those proteins that are distinctively up-regulated.
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References
Allison SD, Vitousek PM (2005) Responses of extracellular enzymes to simple and complex nutrient inputs. Soil Biol Biochem 37:937–944
Allison SD, Weintraub MN, Gartner TB, Waldrop MP (2011) Evolutionary-economic principles as regulators of soil enzyme production and ecosystem. In: Shukla G, Varma A (eds) Soil enzymology, soil biology 22. Springer-Verlag, Berlin, pp. 229–243
Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169
Barber RD (2007) Methanogenesis: ecology. In: Encyclopedia of life sciences. John Wiley and Sons, Ltd. Hoboken, USA. doi:10.1002/9780470015902.a0000475.pub2
Batstone DJ, Keller J, Angelidaki I, Kalyuzhnyi SV, Pavlostathis SG, Rozzi A, Sanders WT, Siegrist H, Vavilin VA (2002) The IWA anaerobic digestion model No 1 (ADM1). Water Sci Technol 45:65–73
Boubaker F, Ridha BC (2008) Implementation of IWA anaerobic digestion model No. 1 (ADM1) for simulating the thermophilic anaerobic co-digestion of olive mill wastewater with olive mill solid waste in a semi-continuous tubular digester. Chem Eng J 141:75–88
Buswell AM and Hatfield DW (1936) Bulletin No. 32, anaerobic fermentations. State of Illinois department of registration and education, division of the state water survey, Urbana, USA
Carle-Urioste JC, Escobar-Vera J, El-Gogary S, Henrique-Silva F, Torigoi E, Crivellaro O, Herrera-Estrella A, El-Dorry H (1997) Cellulase induction in Trichoderma reesei by cellulose requires its own basal expression. J Biol Chem 272:10169–10174
Coleman DJ, Studler MJ, Naleway JJ (2007) A long-wavelength fluorescent substrate for continuous fluorometric determination of cellulase activity: resorufin-β-D-cellobioside. Anal Biochem 371:146–153
Dar SA, Kleerebezem R, Stams AJM, Kuenen JG, Muyzer G (2008) Competition and coexistence of sulfate-reducing bacteria, acetogens and methanogens in a lab-scale anaerobic bioreactor as affected by changing substrate to sulfate ratio. Appl Microbiol Biotechnol 78:1045–1055
Demirjian DC, Moris-Varas F, Cassidy CS (2001) Enzymes from extremophiles. Curr Opin Chem Biol 5:144–151
Drosg B, Braun R, Bochmann G, Al Saedi T (2013) Analysis and characterization of biogas feedstocks. In: Wellinger A, Murphy J, Baxter D (eds) The biogas handbook: science, production and applications. Woodhead Publishing Ltd, Sawston, p. 76
Egli T, Zinn M (2003) The concept of multiple-nutrient-limited growth of microorganisms and its application in biotechnological processes. Biotechnol Adv 22:35–43
Elbeshbishy E, Nakhla G (2012) Batch anaerobic co-digestion of proteins and carbohydrates. Bioresource Technol 116:170–178
Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols handbook. Humana press, New York, pp. 571–607
Geisseler D, Horwath WR (2008) Regulation of extracellular protease activity in soil in response to different sources and concentrations of nitrogen and carbon. Soil Biol Biochem 40:3040–3048
Gurung N, Ray S, Bose S, Rai V (2013) A broader view: microbial enzymes and their relevance in industries, medicine and beyond. Biomed Res Int, vol 2013, article ID 329191
Han SO, Cho HY, Yukawa H, Inui M, Doi RH (2004) Regulation of expression of cellulosomes and noncellulosomal (hemi) cellulolytic enzymes in Clostridium cellulovorans during growth on different carbon sources. J Bacteriol 186:4218–4227
Handelsman J, Rondon MR, Brady SF, Clardy J, Goodman RM (1998) Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem Biol 5:245–249
Johnson EA, Madia A, Demain AL (1981) Chemically defined minimal medium for growth of the anaerobic cellulolytic thermophile Clostridium thermocellum. Appl Environ Microbiol 41:1060–1062
Kim IJ, Lee HJ, Choi I-G, Kim KH (2014) Synergistic proteins for the enhanced enzymatic hydrolysis of cellulose by cellulase. Appl Microbiol Biotechnol 98:8469–8480
Kirk O, Borchert TV, Fuglsang CC (2002) Industrial enzyme applications. Curr Opin Biotech 13:345–351
Kopečný J, Hodrová B (1997) The effect of yellow affinity substance on cellulases of Ruminococcus flavefaciens. Lett Appl Microbiol 25:191–196
Kuhad RC, Gupta R, Singh A (2011) Microbial cellulases and their industrial applications. Enz Res, vol 2011, article ID 280696
Langer M, Gabor EM, Liebeton K, Meurer G, Niehaus F, Schulze R, Eck J, Lorenz P (2006) Metagenomics: an inexhaustible access to nature’s diversity. Biotechnol J 1:815–821
Li S, Yang X, Yang S, Zhu M, Wang X (2012) Technology prospecting on enzymes: application, marketing and engineering. Comput Struct Biotechnol J 2:1–11
Ljungdahl LG, Pettersson B, Eriksson KE, Wiegel J (1983) A yellow affinity substance involved in the cellulolytic system of Clostridium thermocellum. Curr Microbiol 9:195–199
Lorenz P, Eck J (2005) Metagenomics and industrial applications. Nat Rev Micro 3:510–516
Mienda BS, Yahya A, Galadima IA, Shamsir MS (2014) An overview of microbial proteases for industrial applications. Res J Pharm Biol Chem Sci 5:388–396
Mould FL, Morgan R, Kliem KE, Krystallidou E (2005) A review and simplification of the in vitro incubation medium. Anim Feed Sci Tech 123:155–172
Niehaus F, Bertoldo C, Kähler M, Antranikian G (1999) Extremophiles as a source of novel enzymes for industrial application. Appl Microbiol Biotechnol 5:711–729
Nordell E, Moestedt J, Karlsson M (2011) Biogas producing laboratory reactor. SE Patent 1150954–4
Parawira W (2012) Enzyme research and applications in biotechnological intensification of biogas production. Crit Rev Biotechnol 32:172–186
Polizzi KM, Bommarius AS, Broering JM, Chaparro-Riggers JF (2007) Stability of biocatalysts. Curr Opin Chem Biol 11:220–225
Rappe MS, Giovannoni SJ (2003) The uncultured microbial majority. Annu Rev Microbiol 57:369–394
Rinke C, Schwientek P, Sczyrba A, Ivanova NN, Anderson IJ, Cheng J-F, Darling A, Malfatti S, Swan BK, Gies EA, Dodsworth JA, Hedlund BP, Tsiamis G, Sievert SM, Liu W-T, Eisen JA, Hallam SJ, Kyrpides NC, Stepanauskas R, Rubin EM, Hugenholtz P, Woyke T (2013) Insights into the phylogeny and coding potential of microbial dark matter. Nature 499:431–437
Rogowska-Wrzesinska A, Le Bihan M-C, Thaysen-Andersen M, Roepstorff P (2013) 2D gels still have a niche in proteomics. J Proteome 88:4–13
Rothschild LJ, Mancinelli RL (2001) Life in extreme environments. Nature 409:1092–1101
Schloss PD, Handelsman J (2003) Biotechnological prospects from metagenomics. Curr Opin Biotechnol 14:303–310
Sharpton TJ (2014) An introduction to the analysis of shotgun metagenomic data. Front Plant Sci 5:209
Shink B (2002) Synergistic interactions in the microbial world. A Van Leeuw J Microb 81:257–261
Singhania RR, Patel AK, Sukumaran RK, Larroche C, Pandey A (2013) Role and significance of β- glucosidases in the hydrolysis of cellulose for bioethanol production. Biores Technol 127:500–507
SS-EN 12176 (1998) Characterization of sludges—determination of pH. STD-23050. Swedish Standards Institute, Stockholm, Sweden
SS-EN ISO 9963–2 (1994) Water quality—determination of alkalinity—part 2: determination of carbonate alkalinity. STD-18780. Swedish Standards Institute, Stockholm, Sweden
Staley JT, Konopka A (1985) Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol 39:369–394
Sternberg D, Mandels GR (1979) Induction of cellulolytic enzymes in Trichoderma reesei by sophorose. J Bacteriol 139:761–769
Stewart EJ (2012) Growing unculturable bacteria. J Bacteriol 194:4151–4160
Streit WR, Schmitz RA (2004) Metagenomics—the key to the uncultured microbes. Curr Opin Microbiol 7(5):492–498
Sweeney MD, Xu F (2012) Biomass converting enzymes as industrial biocatalysts for fuels and chemicals: recent developments. Catalysts 2:244–263
Torsvik V, Øvreås L, Thingstad TF (2002) Prokaryotic diversity—magnitude, dynamics, and controlling factors. Science 296:1064–1066
Wilmes P, Bond PL (2004) The application of two-dimensional polyacrylamide gel electrophoresis and downstream analyses to a mixed community of prokaryotic microorganisms. Environ Microbiol 6:911–920
Zinder SH (1984) Microbiology of anaerobic conversion of organic wastes to methane: recent developments. ASM News 50:294–298
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The technical support from Tekniska Verken i Linköping for running and maintaining the bioreactors is greatly appreciated.
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This work was financially supported by the Swedish Research Council (grant to Martin Karlsson, number 621-2009-4150) and InZymes Biotech AB.
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Martin Karlsson is affiliated to both InZymes Biotech AB and Linköping University.
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This article does not contain any studies with human participants or animals performed by any of the authors.
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Jutta Speda and Mikaela A. Johansson contributed equally to the manuscript.
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Speda, J., Johansson, M.A., Jonsson, BH. et al. Applying theories of microbial metabolism for induction of targeted enzyme activity in a methanogenic microbial community at a metabolic steady state. Appl Microbiol Biotechnol 100, 7989–8002 (2016). https://doi.org/10.1007/s00253-016-7547-z
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DOI: https://doi.org/10.1007/s00253-016-7547-z