Production of Gloeophyllum trabeum Endoglucanase Cel12A in Nicotiana benthamiana for Cellulose Degradation.

Kyoung Rok Geem,Dong Wook Lee,Younho Song,Inhwan Hwang,Hyeun-Jong Bae

doi:10.3389/fpls.2021.696199

Abstract

Lignocellulosic biomass from plants has been used as a biofuel source and the potent acidic endoglucanase GtCel12A has been isolated from Gloeophyllum trabeum, a filamentous fungus. In this study, we established a plant-based platform for the production of active GtCel12A fused to family 3 cellulose-binding module (CBM3). We used the signal sequence of binding immunoglobulin protein (BiP) and the endoplasmic reticulum (ER) retention signal for the accumulation of the produced GtCel12A in the ER. To achieve enhanced enzyme expression, we incorporated the M-domain of the human receptor-type tyrosine-protein phosphatase C into the construct. In addition, to enable the removal of N-terminal domains that are not necessary after protein expression, we further incorporated the cleavage site of Brachypodium distachyon small ubiquitin-like modifier. The GtCel12A-CBM3 fusion protein produced in the leaves of Nicotiana benthamiana exhibited not only high solubility but also efficient endoglucanase activity on the carboxymethyl cellulose substrate as determined by 3,5-dinitrosalicylic acid assay. The endoglucanase activity of GtCel12A-CBM3 was maintained even when immobilized on microcrystalline cellulose beads. Taken together, these results indicate that GtCel12A endoglucanase produced in plants might be used to provide monomeric sugars from lignocellulosic biomass for bioethanol production.

Highlights

Due to the energy crisis caused by the gradual depletion of fossil fuel reserves and the accumulation of greenhouse gases such as carbon dioxide and methane, caused by high consumption of fossil fuels, the demand for alternative renewable energy sources is increasing (Jeswani et al, 2020; Kumar et al, 2020; Liu et al, 2021)
Lignocellulose is mainly composed of carbohydrate polymers, such as cellulose and hemicellulose, and an aromatic polymer, lignin
To generate the binding immunoglobulin protein (BiP)-M-bdSUMOGtCel12A-CBM3-HDEL construct, we digested with XmaI and Acc65I the pCambia1300 plant expression vector (Komori et al, 2007; Razzak et al, 2020), containing the sequences encoding for the BiP signal sequence, M domain of the human receptor-type tyrosine-protein phosphatase C, SUMO domain, and CBM3HDEL, and ligated the GtCel12A sequence into it that was digested with the same restriction endonucleases

Summary

Introduction

Due to the energy crisis caused by the gradual depletion of fossil fuel reserves and the accumulation of greenhouse gases such as carbon dioxide and methane, caused by high consumption of fossil fuels, the demand for alternative renewable energy sources is increasing (Jeswani et al, 2020; Kumar et al, 2020; Liu et al, 2021). Among the several types of biomass resources, plant-derived lignocellulosic biomass is the most abundant raw material (Fatma et al, 2018; Toor et al, 2020). During lignocellulosic biomass-derived biofuel production, the efficient conversion of carbohydrate polymers into monomeric sugars, later used for ethanol production through fermentation, are of utmost importance for cost-efficiency (Liu et al, 2018; Ali et al, 2020; Barbosa et al, 2020). The production costs of cellulase, an enzyme converting carbohydrate polymers into monomeric sugars, account for ∼40% of the total lignocellulosebased bioethanol production cost (Behera and Ray, 2016). The development of a platform for the cost-efficient production of cellulase harboring high stability and activity might provide a promising avenue in the bioethanol industry

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