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Methods development for metaproteomics-guided bioprospecting of novel enzymes
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
2016 (English)Doctoral 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.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2016. , p. 72
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1819
National Category
Chemical Sciences Microbiology Bioinformatics and Systems Biology Biochemistry and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:liu:diva-133206DOI: 10.3384/diss.diva-133206ISBN: 9789176856109 (print)OAI: oai:DiVA.org:liu-133206DiVA, id: diva2:1056314
Public defence
2017-02-03, Planck, Fysikhuset, Campus Valla, Linköping, 10:15 (English)
Opponent
Supervisors
Available from: 2016-12-14 Created: 2016-12-14 Last updated: 2017-02-06Bibliographically approved
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, p. 7989-8002Article 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
Keywords
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: 2018-05-15
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, p. 23-36Article 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
Keywords
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: 2018-05-15Bibliographically approved

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