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Title: Expression of Aspergillus niger CAZymes is determined by compositional changes in wheat straw generated by hydrothermal or ionic liquid pretreatments

Abstract

The capacity of fungi, such as Aspergillus niger, to degrade lignocellulose is harnessed in biotechnology to generate biofuels and high-value compounds from renewable feedstocks. Most feedstocks are currently pretreated to increase enzymatic digestibility: improving our understanding of the transcriptomic responses of fungi to pretreated lignocellulosic substrates could help to improve the mix of activities and reduce the production costs of commercial lignocellulose saccharifying cocktails. We investigated the responses of A. niger to untreated, ionic liquid and hydrothermally pretreated wheat straw over a 5-day time course using RNA-seq and targeted proteomics. The ionic liquid pretreatment altered the cellulose crystallinity while retaining more of the hemicellulosic sugars than the hydrothermal pretreatment. Ionic liquid pretreatment of straw led to a dynamic induction and repression of genes, which was correlated with the higher levels of pentose sugars saccharified from the ionic liquid-pretreated straw. Hydrothermal pretreatment of straw led to reduced levels of transcripts of genes encoding carbohydrate-active enzymes as well as the derived proteins and enzyme activities. Both pretreatments abolished the expression of a large set of genes encoding pectinolytic enzymes. These reduced levels could be explained by the removal of parts of the lignocellulose by the hydrothermal pretreatment. The time course also facilitatedmore » identification of temporally limited gene induction patterns. The presented transcriptomic and biochemical datasets demonstrate that pretreatments caused modifications of the lignocellulose, to both specific structural features as well as the organisation of the overall lignocellulosic structure, that determined A. niger transcript levels. The experimental setup allowed reliable detection of substrate-specific gene expression patterns as well as hitherto non-expressed genes. Our data suggest beneficial effects of using untreated and IL-pretreated straw, but not HT-pretreated straw, as feedstock for CAZyme production.« less

Authors:
 [1];  [2];  [3];  [3];  [3];  [3];  [4];  [4];  [4];  [4];  [4];  [5];  [5];  [6];  [7];  [3];  [4];  [3];  [5];  [3]
  1. Univ. of Nottingham, Nottingham (United Kingdom); Utrecht Univ., Utrecht (The Netherlands)
  2. Univ. of Nottingham, Nottingham (United Kingdom); Univ. of Manchester, Manchester (United Kingdom)
  3. Univ. of Nottingham, Nottingham (United Kingdom)
  4. U.S. Dept. of Energy Joint Genome Institute, Walnut Creek, CA (United States)
  5. Joint BioEnergy Institute, Emeryville, CA (United States)
  6. Univ. of Nottingham, Nottingham (United Kingdom); Public Health England, Salisbury (United Kingdom)
  7. Univ. of Nottingham, Nottingham (United Kingdom); Sorbonne Univ., Paris (France)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1379724
Grant/Contract Number:
AC02-05CH11231
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 10; Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; Aspergillus niger; Lignocellulose; Ionic liquid and hydrothermal pretreatments; Straw; Transcriptomic responses; CAZy; Hemicellulose; RNA-seq; Targeted proteomics

Citation Formats

Daly, Paul, van Munster, Jolanda M., Blythe, Martin J., Ibbett, Roger, Kokolski, Matt, Gaddipati, Sanyasi, Lindquist, Erika, Singan, Vasanth R., Barry, Kerrie W., Lipzen, Anna, Ngan, Chew Yee, Petzold, Christopher J., Chan, Leanne Jade G., Pullan, Steven T., Delmas, Stephane, Waldron, Paul R., Grigoriev, Igor V., Tucker, Gregory A., Simmons, Blake A., and Archer, David B.. Expression of Aspergillus niger CAZymes is determined by compositional changes in wheat straw generated by hydrothermal or ionic liquid pretreatments. United States: N. p., 2017. Web. doi:10.1186/s13068-017-0700-9.
Daly, Paul, van Munster, Jolanda M., Blythe, Martin J., Ibbett, Roger, Kokolski, Matt, Gaddipati, Sanyasi, Lindquist, Erika, Singan, Vasanth R., Barry, Kerrie W., Lipzen, Anna, Ngan, Chew Yee, Petzold, Christopher J., Chan, Leanne Jade G., Pullan, Steven T., Delmas, Stephane, Waldron, Paul R., Grigoriev, Igor V., Tucker, Gregory A., Simmons, Blake A., & Archer, David B.. Expression of Aspergillus niger CAZymes is determined by compositional changes in wheat straw generated by hydrothermal or ionic liquid pretreatments. United States. doi:10.1186/s13068-017-0700-9.
Daly, Paul, van Munster, Jolanda M., Blythe, Martin J., Ibbett, Roger, Kokolski, Matt, Gaddipati, Sanyasi, Lindquist, Erika, Singan, Vasanth R., Barry, Kerrie W., Lipzen, Anna, Ngan, Chew Yee, Petzold, Christopher J., Chan, Leanne Jade G., Pullan, Steven T., Delmas, Stephane, Waldron, Paul R., Grigoriev, Igor V., Tucker, Gregory A., Simmons, Blake A., and Archer, David B.. Tue . "Expression of Aspergillus niger CAZymes is determined by compositional changes in wheat straw generated by hydrothermal or ionic liquid pretreatments". United States. doi:10.1186/s13068-017-0700-9. https://www.osti.gov/servlets/purl/1379724.
@article{osti_1379724,
title = {Expression of Aspergillus niger CAZymes is determined by compositional changes in wheat straw generated by hydrothermal or ionic liquid pretreatments},
author = {Daly, Paul and van Munster, Jolanda M. and Blythe, Martin J. and Ibbett, Roger and Kokolski, Matt and Gaddipati, Sanyasi and Lindquist, Erika and Singan, Vasanth R. and Barry, Kerrie W. and Lipzen, Anna and Ngan, Chew Yee and Petzold, Christopher J. and Chan, Leanne Jade G. and Pullan, Steven T. and Delmas, Stephane and Waldron, Paul R. and Grigoriev, Igor V. and Tucker, Gregory A. and Simmons, Blake A. and Archer, David B.},
abstractNote = {The capacity of fungi, such as Aspergillus niger, to degrade lignocellulose is harnessed in biotechnology to generate biofuels and high-value compounds from renewable feedstocks. Most feedstocks are currently pretreated to increase enzymatic digestibility: improving our understanding of the transcriptomic responses of fungi to pretreated lignocellulosic substrates could help to improve the mix of activities and reduce the production costs of commercial lignocellulose saccharifying cocktails. We investigated the responses of A. niger to untreated, ionic liquid and hydrothermally pretreated wheat straw over a 5-day time course using RNA-seq and targeted proteomics. The ionic liquid pretreatment altered the cellulose crystallinity while retaining more of the hemicellulosic sugars than the hydrothermal pretreatment. Ionic liquid pretreatment of straw led to a dynamic induction and repression of genes, which was correlated with the higher levels of pentose sugars saccharified from the ionic liquid-pretreated straw. Hydrothermal pretreatment of straw led to reduced levels of transcripts of genes encoding carbohydrate-active enzymes as well as the derived proteins and enzyme activities. Both pretreatments abolished the expression of a large set of genes encoding pectinolytic enzymes. These reduced levels could be explained by the removal of parts of the lignocellulose by the hydrothermal pretreatment. The time course also facilitated identification of temporally limited gene induction patterns. The presented transcriptomic and biochemical datasets demonstrate that pretreatments caused modifications of the lignocellulose, to both specific structural features as well as the organisation of the overall lignocellulosic structure, that determined A. niger transcript levels. The experimental setup allowed reliable detection of substrate-specific gene expression patterns as well as hitherto non-expressed genes. Our data suggest beneficial effects of using untreated and IL-pretreated straw, but not HT-pretreated straw, as feedstock for CAZyme production.},
doi = {10.1186/s13068-017-0700-9},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 10,
place = {United States},
year = {Tue Feb 07 00:00:00 EST 2017},
month = {Tue Feb 07 00:00:00 EST 2017}
}

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  • Efficient deconstruction of plant biomass is a major barrier to the development of viable lignocellulosic biofuels. Pretreatment with ionic liquids reduces lignocellulose recalcitrance to enzymatic hydrolysis, increasing yields of sugars for conversion into biofuels. However, commercial cellulases are not compatible with many ionic liquids, necessitating extensive water washing of pretreated biomass prior to hydrolysis. To circumvent this issue, previous research has demonstrated that several thermophilic bacterial cellulases can efficiently deconstruct lignocellulose in the presence of the ionic liquid, 1-ethyl-3-methylimadizolium acetate. As promising as these enzymes are, they would need to be produced at high titer in an industrial enzyme productionmore » host before they could be considered a viable alternative to current commercial cellulases. Aspergillus Niger has been used to produce high titers of secreted enzymes in industry and therefore, we assessed the potential of this organism to be used as an expression host for these ionic liquid-tolerant cellulases. We demonstrated that 29 of these cellulases were expressed at detectable levels in a wild-type strain of A. Niger, indicating a basic level of compatibility and potential to be produced at high levels in a host engineered to produce high titers of enzymes. We then profiled one of these enzymes in detail, the β-glucosidase A5IL97, and compared versions expressed in both A. Niger and Escherichia coli. Finally, this comparison revealed the enzymatic activity of A5IL97 purified from E. coli and A. Niger is equivalent, suggesting that A. Niger could be an excellent enzyme production host for enzymes originally characterized in E. coli, facilitating the transition from the laboratory to industry.« less
  • Efficient deconstruction of plant biomass is a major barrier to the development of viable lignocellulosic biofuels. Pretreatment with ionic liquids reduces lignocellulose recalcitrance to enzymatic hydrolysis, increasing yields of sugars for conversion into biofuels. However, commercial cellulases are not compatible with many ionic liquids, necessitating extensive water washing of pretreated biomass prior to hydrolysis. To circumvent this issue, previous research has demonstrated that several thermophilic bacterial cellulases can efficiently deconstruct lignocellulose in the presence of the ionic liquid, 1-ethyl-3-methylimadizolium acetate. As promising as these enzymes are, they would need to be produced at high titer in an industrial enzyme productionmore » host before they could be considered a viable alternative to current commercial cellulases. Aspergillus Niger has been used to produce high titers of secreted enzymes in industry and therefore, we assessed the potential of this organism to be used as an expression host for these ionic liquid-tolerant cellulases. We demonstrated that 29 of these cellulases were expressed at detectable levels in a wild-type strain of A. Niger, indicating a basic level of compatibility and potential to be produced at high levels in a host engineered to produce high titers of enzymes. We then profiled one of these enzymes in detail, the β-glucosidase A5IL97, and compared versions expressed in both A. Niger and Escherichia coli. Finally, this comparison revealed the enzymatic activity of A5IL97 purified from E. coli and A. Niger is equivalent, suggesting that A. Niger could be an excellent enzyme production host for enzymes originally characterized in E. coli, facilitating the transition from the laboratory to industry.« less
  • Two types of processes have been applied for the pretreatment of lignocellulosic materials in order to render them easily degradable by the cellulase of Trichoderma viride. They were compared at different temperatures, with regard both to the residual dry mass and the improvement in accessibility to the enzyme complex. The latter was measured in terms of glucose liberated, which was quantified by HPLC. Hydrothermolysis proved more effective than the organosolv process for the pretreatment of wheat straw, and vice versa for poplar wood. In terms of the percentage of glucan enzymatically converted to glucose, yields up to 90% could bemore » achieved. 16 references.« less
  • The plant storage carbohydrate inulin represents an attractive biomass feedstock for fueling industrial scale bioconversion processes due to its low cost, ability for cultivation on arid and semi-arid lands, and amenability to consolidated bioprocessing applications. As a result, increasing efforts are emerging towards engineering industrially relevant microorganisms, such as yeast, to efficiently ferment inulin into high value fuels and chemicals. Although some strains of the industrially relevant yeast model Saccharomyces cerevisiae can naturally ferment inulin, the efficiency of this process is often supplemented through expression of exogenous inulinase enzymes that externally convert inulin into its more easily fermentable component monomericmore » sugars. Here, the effects of overexpressing the Aspergillus niger InuA inulinase enzyme in an S. cerevisiae strain incapable of endogenously fermenting inulin were evaluated to determine their impact on growth. Expression of the A. niger InuA inulinase enzyme permitted growth on otherwise intractable inulin substrates from both Dahlia tubers and Chicory root. Despite being in the top 10 secreted proteins, growth on inulin was not observed until 120 h post-inoculation and required the addition of 0.1 g fructose/l to initiate enzyme production in the absence of endogenous inulinase activity. High temperature/pressure pre-treatment of inulin prior to fermentation decreased this time to 24 h and removed the need for fructose addition. The pre-growth lag time on untreated inulin was attributed primarily to low enzymatic efficiency, with a maximum value of 0.13 0.02 U InuA/ml observed prior to the peak culture density of 2.65 0.03 g/l. Nevertheless, a minimum excreted enzymatic activity level of only 0.03 U InuA/ml was found to be required for sustained growth under laboratory conditions, suggesting that future metabolic engineering strategies can likely redirect carbon flow away from inulinase production and reorient it towards product production or cellular growth in order to optimize strain development.« less