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  • 1.
    Speda, Jutta
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Methods development for metaproteomics-guided bioprospecting of novel enzymes2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Industrial biotechnology has been announced by several organizations and governments as a key enabling technology for the enhanced economic growth in a low-carbon and knowledge-based bioeconomy. An important goal to promote an environment friendly and sustainable industrial biotechnology is the discovery of new enzymes.

    To date, almost all enzymes used in industry have been discovered by pure culturing of microorganisms, however, it is known that less than 1% of all microorganisms can be obtained in pure cultures. The remaining majority of microorganisms is only viable by close biological interactions provided in microbial communities and is not available for enzyme discovery using the classical pure culture approaches. The investigation of microbial communities, which can be viewed as metaorganisms, has been enabled during the last two decades by refining established methods for the analysis of genes, mRNA or proteins and are called metagenomics, metatranscriptomics and metaproteomics, respectively. To date, these techniques have mostly been used in the field of microbial ecology for the understanding of the composition, function and metabolism of microbial communities but not for the purpose of bioprospecting for novel enzymes. Identification of genes that code for possible enzyme candidates is hindered, due to the fact that 30-40% of the sequenced metagenomes contain genes coding for unidentified proteins. Additionally, the -omics techniques generate large amounts of data that need to be analyzed and the outcome of the analysis does not necessarily lead to the discovery of novel applicable enzymes.

    The work presented in this thesis describes the establishment of the necessary conditions for a metaproteomics-based method that allows for a straightforward and targeted identification of novel enzymes with desired activity from microbial communities. The approach provides a valuable alternative to the incomplete and inefficient analysis of non-targeting data and laborious workflow, which is typically generated by the established meta-omics techniques. In developing the methods presented in this thesis, microbial communities in constructed environments were established, which allowed for the controlled expression of extracellular hydrolytic enzymes under defined conditions. By combination and modulation of advanced metaproteomics and metagenomics techniques, we were able to directly identify the enzymes and the corresponding gene sequences of several cellulolytic enzymes as a first example for the feasibility of this approach.

    List of papers
    1. Applying theories of microbial metabolism for induction of targeted enzyme activity in a methanogenic microbial community at a metabolic steady state
    Open this publication in new window or tab >>Applying theories of microbial metabolism for induction of targeted enzyme activity in a methanogenic microbial community at a metabolic steady state
    2016 (English)In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 100, no 18, 7989-8002 p.Article in journal (Refereed) Published
    Abstract [en]

    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.

    Place, publisher, year, edition, pages
    Springer, 2016
    Keyword
    Microbial community; Enzyme discovery; Metaproteomics; Biogas; Cellulase; Protease
    National Category
    Microbiology
    Identifiers
    urn:nbn:se:liu:diva-131888 (URN)10.1007/s00253-016-7547-z (DOI)000382008000017 ()27115757 (PubMedID)
    Note

    Funding Agencies|Swedish Research Council [621-2009-4150]; InZymes Biotech AB

    Available from: 2016-10-13 Created: 2016-10-11 Last updated: 2017-11-29
    2. Assessment of sample preparation methods for metaproteomics of extracellular proteins
    Open this publication in new window or tab >>Assessment of sample preparation methods for metaproteomics of extracellular proteins
    2017 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 516, 23-36 p.Article in journal (Refereed) Published
    Abstract [en]

    Enzyme discovery in individual strains of microorganisms is compromised by the limitations of pure culturing. In principle, metaproteomics allows for fractionation and study of different parts of the protein complement but has hitherto mainly been used to identify intracellular proteins. However, the extracellular environment is also expected to comprise a wealth of information regarding important proteins. An absolute requirement for metaproteomic studies of protein expression, and irrespective of downstream methods for analysis, is that sample preparation methods provide clean, concentrated and representative samples of the protein complement. A battery of methods for concentration, extraction, precipitation and resolubilization of proteins in the extracellular environment of a constructed microbial community was assessed by means of 2D gel electrophoresis and image analysis to elucidate whether it is possible to make the extracellular protein complement available for metaproteomic analysis. Most methods failed to provide pure samples and therefore negatively influenced protein gel migration and gel background clarity. However, one direct precipitation method (TCA-DOC/acetone) and one extraction/precipitation method (phenol/methanol) provided complementary high quality 2D gels that allowed for high spot detection ability and thereby also spot detection of less abundant extracellular proteins.

    Place, publisher, year, edition, pages
    Elsevier, 2017
    Keyword
    Enzyme discovery, Microbial community, Metaproteome, Extracellular, Sample preparation, 2D gel electrophoresis
    National Category
    Analytical Chemistry Biocatalysis and Enzyme Technology
    Identifiers
    urn:nbn:se:liu:diva-132902 (URN)10.1016/j.ab.2016.10.008 (DOI)000388056800005 ()27742212 (PubMedID)
    Funder
    Swedish Research Council, 621-2009-4150
    Note

    Funding agencies: Swedish Research Council [621-2009-4150]; Tekniska Verken i Linkoping AB; InZymes Biotech AB

    Available from: 2016-12-01 Created: 2016-12-01 Last updated: 2017-11-29Bibliographically approved
  • 2.
    Speda, Jutta
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Johansson, Mikaela A.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. InZymes Biotech AB, Linköping, Sweden.
    Assessment of sample preparation methods for metaproteomics of extracellular proteins2017In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 516, 23-36 p.Article in journal (Refereed)
    Abstract [en]

    Enzyme discovery in individual strains of microorganisms is compromised by the limitations of pure culturing. In principle, metaproteomics allows for fractionation and study of different parts of the protein complement but has hitherto mainly been used to identify intracellular proteins. However, the extracellular environment is also expected to comprise a wealth of information regarding important proteins. An absolute requirement for metaproteomic studies of protein expression, and irrespective of downstream methods for analysis, is that sample preparation methods provide clean, concentrated and representative samples of the protein complement. A battery of methods for concentration, extraction, precipitation and resolubilization of proteins in the extracellular environment of a constructed microbial community was assessed by means of 2D gel electrophoresis and image analysis to elucidate whether it is possible to make the extracellular protein complement available for metaproteomic analysis. Most methods failed to provide pure samples and therefore negatively influenced protein gel migration and gel background clarity. However, one direct precipitation method (TCA-DOC/acetone) and one extraction/precipitation method (phenol/methanol) provided complementary high quality 2D gels that allowed for high spot detection ability and thereby also spot detection of less abundant extracellular proteins.

  • 3.
    Speda, Jutta
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Johansson, Mikaela
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. InZymes Biotech AB, Gjuterigatan 1B, S-58273 Linkoping, Sweden.
    Applying theories of microbial metabolism for induction of targeted enzyme activity in a methanogenic microbial community at a metabolic steady state2016In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 100, no 18, 7989-8002 p.Article in journal (Refereed)
    Abstract [en]

    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.

  • 4.
    Speda, Jutta
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Johansson, Mikaela
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Odnell, Anna
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. Karshult Municipal Waste Water Treatment Plant, Sweden.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. InZymes Biotech AB, Gjuterigatan 1B, S-58273 Linkoping, Sweden.
    Enhanced biomethane production rate and yield from lignocellulosic ensiled forage ley by in situ anaerobic digestion treatment with endogenous cellulolytic enzymes2017In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 10, 129Article in journal (Refereed)
    Abstract [en]

    Background: Enzymatic treatment of lignocellulosic material for increased biogas production has so far focused on pretreatment methods. However, often combinations of enzymes and different physicochemical treatments are necessary to achieve a desired effect. This need for additional energy and chemicals compromises the rationale of using enzymes for low energy treatment to promote biogas production. Therefore, simpler and less energy intensive in situ anaerobic digester treatment with enzymes is desirable. However, investigations in which exogenous enzymes are added to treat the material in situ have shown mixed success, possibly because the enzymes used originated from organisms not evolutionarily adapted to the environment of anaerobic digesters. In this study, to examine the effect of enzymes endogenous to methanogenic microbial communities, cellulolytic enzymes were instead overproduced and collected from a dedicated methanogenic microbial community. By this approach, a solution with very high endogenous microbial cellulolytic activity was produced and tested for the effect on biogas production from lignocellulose by in situ anaerobic digester treatment. Results: Addition of enzymes, endogenous to the environment of a mixed methanogenic microbial community, to the anaerobic digestion of ensiled forage ley resulted in significantly increased rate and yield of biomethane production. The enzyme solution had an instant effect on more readily available cellulosic material. More importantly, the induced enzyme solution also affected the biogas production rate from less accessible cellulosic material in a second slower phase of lignocellulose digestion. Notably, this effect was maintained throughout the experiment to completely digested lignocellulosic substrate. Conclusions: The induced enzyme solution collected from a microbial methanogenic community contained enzymes that were apparently active and stable in the environment of anaerobic digestion. The enzymatic activity had a profound effect on the biogas production rate and yield, comparable with the results of many pretreatment methods. Thus, application of such enzymes could enable efficient low energy in situ anaerobic digester treatment for increased biomethane production from lignocellulosic material.

  • 5.
    Speda, Jutta
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. InZymes Biotech AB, Gjuterigatan 1B, S-58273 Linkoping, Sweden.
    Metaproteomics-guided selection of targeted enzymes for bioprospecting of mixed microbial communities2017In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 10, 128Article in journal (Refereed)
    Abstract [en]

    Background: Hitherto, the main goal of metaproteomic analyses has been to characterize the functional role of particular microorganisms in the microbial ecology of various microbial communities. Recently, it has been suggested that metaproteomics could be used for bioprospecting microbial communities to query for the most active enzymes to improve the selection process of industrially relevant enzymes. In the present study, to reduce the complexity of metaproteomic samples for targeted bioprospecting of novel enzymes, a microbial community capable of producing cellulases was maintained on a chemically defined medium in an enzyme suppressed metabolic steady state. From this state, it was possible to specifically and distinctively induce the desired cellulolytic activity. The extracellular fraction of the protein complement of the induced sample could thereby be purified and compared to a non-induced sample of the same community by differential gel electrophoresis to discriminate between constitutively expressed proteins and proteins upregulated in response to the inducing substance. Results: Using the applied approach, downstream analysis by mass spectrometry could be limited to only proteins recognized as upregulated in the cellulase-induced sample. Of 39 selected proteins, the majority were found to be linked to the need to degrade, take up, and metabolize cellulose. In addition, 28 (72%) of the proteins were non-cytosolic and 17 (44%) were annotated as carbohydrate-active enzymes. The results demonstrated both the applicability of the proposed approach for identifying extracellular proteins and guiding the selection of proteins toward those specifically upregulated and targeted by the enzyme inducing substance. Further, because identification of interesting proteins was based on the regulation of enzyme expression in response to a need to hydrolyze and utilize a specific substance, other unexpected enzyme activities were able to be identified. Conclusions: The described approach created the conditions necessary to be able to select relevant extracellular enzymes that were extracted from the enzyme-induced microbial community. However, for the purpose of bioprospecting for enzymes to clone, produce, and characterize for practical applications, it was concluded that identification against public databases was not sufficient to identify the correct gene or protein sequence for cloning of the identified novel enzymes.

  • 6.
    Speda, Jutta
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology. Rational Enzyme Mining AB, Linköping, Sweden.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology. Rational Enzyme Mining AB, Linköping, Sweden.
    Induced differential metaproteomics for the identification of cellulases in a methanogenic microbial community2013In: BioMicroWorld 2013: Book of Abstracts, 2013, 679- p.Conference paper (Other academic)
    Abstract [en]

    The identification of novel enzymes for use in industrial biotechnology is an important goal in enzyme discovery. The need for novel biocatalysts for sustainable and efficient bioenergy production and the development of new biomaterials especially gives rise to new strategic opportunities of proteomic research. Most industrially relevant enzymes to date have been isolated from pure cultured microorganisms. It is however well established that only a small fraction of all existing microorganisms can be obtained in pure cultures, thus limiting the potential of finding novel enzymes. The possibility to identify valuable enzymes directly from complete microbial communities would therefore potentially give access to a huge number of novel enzyme candidates.

    Metaproteomics, or “the large-scale characterization of the entire protein complement of environmental microbiota at a given point in time” has hitherto mainly been used to understand ecosystem function. In order to reach our goals we have instead used the dynamics of metaproteomics to develop a method based on “induced differential metaproteomics”, in which a desired enzyme activity is induced in a microbial population and compared to a non-induced reference of the very same population. In a first example the goal was to induce, select and identify cellulases from a methanogenic community, maintained in a biogas reactor at a metabolic steady-state in a chemically defined medium.

    Two aliquots were subtracted from the reactor, of which one was treated to induce cellulase activity. At the peak of cellulase activity and biogas production in the induced sample, proteins from the liquid phase of the two samples were prepared for 2D-DIGE of the extra-cellular proteins. Out of several hundred protein spots generated by the microbial community and visible in the 2D-DIGE experiment, 95 could be identified as up-regulated in the induced sample by image analysis, as compared to the references (thus representing potential cellulases). In-gel digestion and tandem mass-spectrometry of located and selected up-regulated proteins revealed that 18 out of 30 proteins could be assigned as cellulases or associated to cellulolytic activity giving a remarkable hit-rate of 60 % and thus demonstrating the feasibility of the approach.

    These cellulases found can be expected to be highly active and stable at the conditions in which they are naturally produced (pH, temp., salinity etc.). A strategic objective of research, both in academia and in thebiotechnology industry, is to identify novel, highly active microbial enzymes that are stable at the different conditions of various industrial applications. Thus, one of our future prospects includes to further employ the described methodology to identify novel enzymes from microbial communities originating from more extreme environments.

  • 7.
    Speda, Jutta
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Induced differential metaproteomics: identification of cellulases in a methanogenic microbial community at mesophilic conditions2014Conference paper (Other academic)
  • 8.
    Speda, Jutta
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Induced differential metaproteomics: identification of thermostable cellulases in a methanogenic microbial community2014Conference paper (Other academic)
    Abstract [en]

    The identification of novel enzymes for use in industrial biotechnology is an important goal in enzyme discovery. Most industrially relevant enzymes to date have been isolated from pure cultured microorganisms. For future discovery of novel enzymes this is however a major bottleneck since it is well established that only a small fraction of all microorganisms can be obtained in pure cultures. The possibility to identify enzymes directly from complete microbial communities would therefore give access to a huge number of novel enzyme candidates.

    Metaproteomics has hitherto mainly been used to understand ecosystem functions. We have instead used the dynamics of proteomics to develop a method based on “induced differential metaproteomics”, by which a desired enzyme activity is induced in a full microbial population and compared to a non-induced reference of the very same population. In a first example the goal was to induce, select and identify cellulases from a thermophilic methanogenic community.

    Out of several hundred detectable proteins in a 2D-DIGE experiment, 24 proteins could be identified as at least two-fold up-regulated upon induction. For some proteins spots, the cellulolytic activity was further validated by activity staining using 2D-zymography. Mass spectrometry analysis revealed that 21 out of the 24 up-regulated proteins are cellulases or associated to cellulolytic activity giving a remarkable hit-rate of 88%. This demonstrates the high efficiency and precision of the method, by which a much wider span of the microbial world can be scanned for novel and targeted enzymes.

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