Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Summary

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

Abstract

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Introduction

Degradation of lignocellulosic biomass and its conversion into liquid fuel has been envisioned as a method to reduce reliance on fossil fuels. One significant hurdle in establishing economically feasible biomass processing systems is in the enzymatic degradation of cellulose and hemicellulose¹. Plant expression systems show great potential for the production of enzymes on an industrial scale. Agricultural systems to harvest plants economically and in volume are already established, as they have been for thousands of years. The production of cellulases in plants is desirable due to factors like the ease of autohydrolysis², and the potential for ma....

Protocol

1. Growth of Wild Type N. tabacum Plants

1. To grow N. tabacum L. cv. Petit Havana SR1 plants on soil, place tobacco seeds on appropriate pots filled with normal potting soil for germination. After 2 weeks, transfer seedlings to individual pots. Incubate plants in a greenhouse with constant 22 °C (dark period) or 25 °C (light period) and 70% relative air humidity. Provide illumination in a 16/8 hr light/dark cycle (e.g. Philips IP65 400 W lamps; 180 μmol·sec^-1·m^-2; λ= 400-700 nm) and water daily.

2. Transient Expression of TrCel5A....

Results

TrCel5A and TrCel5A-mCherry were expressed successfully in tobacco plants using the transient expression system (Figures 1A and 1B). Examination of the expression pattern of the TrCel5A-mCherry fusion protein revealed healthy leaves (Figure 1C), which showed widespread protein expression under green light (Figure 1D).

Extraction of the total soluble proteins was performed on tobacco plants which had leaves that contained the t.......

Discussion

Recombinant expression of cellulases is a field of great interest, due to the desire for more efficient biofuel production systems¹. Plants offer many possibilities for successful cellulase production, with features such as economic harvesting techniques and autohydrolysis methods being very desirable properties for large-scale biofuel production. Further, the use of different localizations for producing proteins allow, if required, posttranslational modifications²⁰. Altering plants to enable consti.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-methylumbelliferyl β-D-cellobioside	SIGMA-ALDRICH	M6018
Acetic acid	ROTH	3738
Acetosyringon (concentrated)	ROTH	6003
Antibiotic - kanamycin	ROTH	T832
Antibiotic - rifampicin	ROTH	4163
Antibiotic - carbenicillin	ROTH	6344
Antibody - rabbit anti His(6) pAb	ROCKLAND	600-401-382
Antibody - goat anti rabbit AP, FC pAb	DIANOVA	111-055-008
Azo-carboxymethyl cellulose	MEGAZYME	S-ACMC
β-cyclodextrin	SIGMA-ALDRICH	C4805
Calcium chloride dihydrate	SIGMA-ALDRICH	C5080
Carboxymethyl cellulose	ROTH	3333.1
Cellulase from Trichoderma reesei ATCC 26921	SIGMA-ALDRICH	C2730
Congo red	SIGMA-ALDRICH	75768
Coomasie brilliant blue G 250	ROTH	9598.2
D(+)-glucose	ROTH	8337
Ethanol	ROTH	K928
Filter paper - Rotilabo blotting paper	ROTH	CL67
NBT/BCIP	SIGMA-ALDRICH	72091
MES buffer	ROTH	4256
Methanol	ROTH	8388
Sodium citrate	ROTH	3580
Sodium dodecylsulfate	SERVA	20765
Sodium hydroxide	ROTH	6771
Soil, Einheitserde classic	PATZER GMBH
Spectrometer - Infinite M200	TECAN
Sucrose	SIGMA-ALDRICH	S-5390
4-Hydroxy benzhydrazide	SIGMA-ALDRICH	H9882
Phosphate buffered saline	ROTH	1058
UV light source - BLAK RAY	UVP
Vibratome - VT1000S	Leica