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In‐depth characterization of Trichoderma reesei cellobiohydrolase TrCel7A produced in Nicotiana benthamiana reveals limitations of cellulase production in plants by host‐specific post‐translational modifications

Plant biotechnology journal, 2020-03, Vol.18 (3), p.631-643 [Peer Reviewed Journal]

2019 The Authors. published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. ;2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. ;COPYRIGHT 2020 John Wiley & Sons, Inc. ;info:eu-repo/semantics/openAccess ;ISSN: 1467-7644 ;EISSN: 1467-7652 ;DOI: 10.1111/pbi.13227 ;PMID: 31373133

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Title:
In‐depth characterization of Trichoderma reesei cellobiohydrolase TrCel7A produced in Nicotiana benthamiana reveals limitations of cellulase production in plants by host‐specific post‐translational modifications
Author: Eerde, André ; Várnai, Anikó ; Jameson, John Kristian ; Paruch, Lisa ; Moen, Anders ; Anonsen, Jan Haug ; Chylenski, Piotr ; Steen, Hege Særvold ; Heldal, Inger ; Bock, Ralph ; Eijsink, Vincent G. H. ; Liu‐Clarke, Jihong
Subjects: Biomass energy ; cellobiohydrolase ; Cellulose 1,4-beta-Cellobiosidase - biosynthesis ; enzyme activity ; Enzymes ; Fungal Proteins - biosynthesis ; Nicotiana - metabolism ; Nicotiana benthamiana ; plant glycosylation ; plant molecular farming ; Plants, Genetically Modified - metabolism ; Post-translational modification ; Protein Processing, Post-Translational ; protein stability ; Recombinant Proteins - biosynthesis ; substrate binding ; Trichoderma - enzymology ; Trichoderma reesei
Is Part Of: Plant biotechnology journal, 2020-03, Vol.18 (3), p.631-643
Description: Summary Sustainable production of biofuels from lignocellulose feedstocks depends on cheap enzymes for degradation of such biomass. Plants offer a safe and cost‐effective production platform for biopharmaceuticals, vaccines and industrial enzymes boosting biomass conversion to biofuels. Production of intact and functional protein is a prerequisite for large‐scale protein production, and extensive host‐specific post‐translational modifications (PTMs) often affect the catalytic properties and stability of recombinant enzymes. Here we investigated the impact of plant PTMs on enzyme performance and stability of the major cellobiohydrolase TrCel7A from Trichoderma reesei, an industrially relevant enzyme. TrCel7A was produced in Nicotiana benthamiana using a vacuum‐based transient expression technology, and this recombinant enzyme (TrCel7Arec) was compared with the native fungal enzyme (TrCel7Anat) in terms of PTMs and catalytic activity on commercial and industrial substrates. We show that the N‐terminal glutamate of TrCel7Arec was correctly processed by N. benthamiana to a pyroglutamate, critical for protein structure, while the linker region of TrCel7Arec was vulnerable to proteolytic digestion during protein production due to the absence of O‐mannosylation in the plant host as compared with the native protein. In general, the purified full‐length TrCel7Arec had 25% lower catalytic activity than TrCel7Anat and impaired substrate‐binding properties, which can be attributed to larger N‐glycans and lack of O‐glycans in TrCel7Arec. All in all, our study reveals that the glycosylation machinery of N. benthamiana needs tailoring to optimize the production of efficient cellulases.
Publisher: England: John Wiley & Sons, Inc
Language: English;Norwegian
Identifier: ISSN: 1467-7644
EISSN: 1467-7652
DOI: 10.1111/pbi.13227
PMID: 31373133
Source: MEDLINE
Wiley Open Access
NORA Norwegian Open Research Archives
ProQuest Central
DOAJ Directory of Open Access Journals

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In‐depth characterization of Trichoderma reesei cellobiohydrolase TrCel7A produced in Nicotiana benthamiana reveals limitations of cellulase production in plants by host‐specific post‐translational modifications

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