Assessment of sample preparation methods for metaproteomics of extracellular proteins

Speda, Jutta; Johansson, Mikaela A.; Carlsson, Uno; Karlsson, Martin

doi:10.1016/j.ab.2016.10.008

Cited by 21 publications

(20 citation statements)

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“…However, because the methanogenic microbial community was maintained on a chemically defined medium under controlled conditions, a metaproteomic analysis of the extracellular proteins was made possible [37]. Recently, using the described approach to preferentially and distinctly induce cellulolytic enzyme activity [29] the resulting clear differences in the protein expression pattern between the induced and the non-induced state were used to pinpoint a number of proteins that were upregulated in response to the need to hydrolyze cellulose [38].…”

Section: Discussionmentioning

confidence: 99%

Enhanced biomethane production rate and yield from lignocellulosic ensiled forage ley by in situ anaerobic digestion treatment with endogenous cellulolytic enzymes

Speda

Johansson

Odnell

et al. 2017

Biotechnol Biofuels

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BackgroundEnzymatic 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.ResultsAddition 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.ConclusionsThe 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.

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Section: Discussionmentioning

confidence: 99%

Enhanced biomethane production rate and yield from lignocellulosic ensiled forage ley by in situ anaerobic digestion treatment with endogenous cellulolytic enzymes

Speda

Johansson

Odnell

et al. 2017

Biotechnol Biofuels

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“…From this enzyme suppressed metabolic steady state, it was found that the desired hydrolytic enzyme activity could be induced at will by replacing the simple nutrients with complex counterparts, e.g., by exchanging glucose for cellulose to induce cellulolytic activity. In addition, it was further found that the extracellular enzymes could be purified to provide 2-D gels of high quality, allowing detection of also less abundant proteins [23]. Recently, the same system was used to produce a mixture of cellulolytic enzymes that were shown to increase the hydrolysis rate of lignocellulosic biomass in biogas batch experiments [24].…”

Section: Introductionmentioning

confidence: 99%

Metaproteomics-guided selection of targeted enzymes for bioprospecting of mixed microbial communities

Speda

Jonsson

Carlsson

et al. 2017

Biotechnol Biofuels

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BackgroundHitherto, 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.ResultsUsing 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.ConclusionsThe 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.

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“…This is of outstanding importance, because it is usually very difficult in meta-omics projects to identify relevant enzymes or target the production of specific enzymes against the large background of all proteins that are collected. In addition, by maintaining the microbial community in a constructed environment on a chemically defined medium, the possibility to collect and analyze the extracellular metaproteome, or the metasecretome, was realized (papers III [119]) Figure 4. Metaproteogenomics-guided bioprospecting for novel enzymes.…”

Section: Metaproteogenomics Is a Rather New Technique In The Integratmentioning

confidence: 99%

“…The upregulated extracellular proteins in the experiment could be visualized and measured by application of sensitive, advanced gel electrophoresis based proteomics techniques (papers III [119], IV and V). The metagenome of the microbial community was sequenced, translated into all six reading-frames and used as a database in a following tandem mass spectrometry experiment for the investigation of the upregulated proteins.…”

Section: Metaproteogenomics Is a Rather New Technique In The Integratmentioning

confidence: 99%

Methods development for metaproteomics-guided bioprospecting of novel enzymes

Speda¹

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Cover: 2-D DIGE gel used for the accurate identification of extracellular cellulolytic enzymes secreted by a methanogenic microbial community. Red protein spots indicate proteins upregulated by the addition of cellulose.During the course of the research underlying this thesis, Jutta Speda was enrolled in Forum Scientium, a multidisciplinary doctoral programme at Linköping University, Sweden. Printed by LiU-Tryck, Linköping, Sweden, 2017 Look deep into nature, and then you will understand everything better. Albert Einstein v AbstractIndustrial 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 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 vi able to directly identify the enzymes and the corresponding gene sequences of several cellulolytic enzymes as a first exam...

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Assessment of sample preparation methods for metaproteomics of extracellular proteins

Cited by 21 publications

References 46 publications

Enhanced biomethane production rate and yield from lignocellulosic ensiled forage ley by in situ anaerobic digestion treatment with endogenous cellulolytic enzymes

Enhanced biomethane production rate and yield from lignocellulosic ensiled forage ley by in situ anaerobic digestion treatment with endogenous cellulolytic enzymes

Metaproteomics-guided selection of targeted enzymes for bioprospecting of mixed microbial communities

Methods development for metaproteomics-guided bioprospecting of novel enzymes

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