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 maximizing the use of lower enzyme levels by increasing the digestibility of plant cell walls¹. Finally, plant systems allow the targeting of recombinant proteins to specific areas of plant cells and are able to posttranslationally modify enzymes where required³.

Nicotiana tabacum (tobacco) is very commonly used as a model organism for heterologous protein expression studies in plants, due to its rapid growth and biomass accumulation features⁴. Transient expression of recombinant proteins is a technique which enables protein production in short time periods⁵, while maintaining flexibility as to the localization and thus posttranslational modifications of the selected proteins, i.e. cellulases. This enables the production of the cellulase(s) for basic analysis, while also laying the groundwork for further expression strategies in plants. Using such a transient expression strategy, an endoglucanase from glycosyl hydrolase family 5, Cel5A, derived from the fungal host Trichoderma reesei (Hypocrea jecorina)⁶ is produced (hereafter referred to as TrCel5A). TrCel5A is a 42 kDa protein which is natively glycosylated and is highly active in hydroylzing cellulose chains⁷.

The transient expression technique described here is based on a relatively commonly used system, infiltrating the plant leaves with Agrobacteria carrying the gene of interest in an appropriate expression vector. To allow for rapid analysis of successful in planta expression, TrCel5A can also be expressed as a fusion protein with mCherry, a monomeric fluorescent protein originally derived from Discosoma sp. protein DsRed⁸, with an additional six Histidine residues (His-tag) fused to the C-terminus (TrCel5A-mCherry). Expression of the heterologous fusion protein, TrCel5A-mCherry, can thus be monitored within the growing plant by using green light to examine mCherry dispersal. If desired, thin sections of the plant material can be examined under green light microscopically to establish the specific localization of the protein. For this work, signal and transit peptides were incorporated in the construct, to localize the heterologous protein export to the endoplasmic reticulum⁹.

To analyze the activity of plant expressed cellulases, including TrCel5A, a number of cellulase activity tests can be run. After the extraction of the total soluble plant proteins, TrCel5A can be partially purified using a thermal incubation technique. Protein size is established using SDS-PAGE followed by Western blotting. Zymography can be used to analyze activity against substrates, e.g. cellulose which has been carboxy-methylated (making carboxymethyl cellulose: CMC) for solubility¹⁰. Cellulase and glucosidase activity can be monitored using the fluorophore 4-methylumbelliferyl (4-MU) associated with β-D-cellobioside (combined: 4-MUC). Another method to assess endoglucanase activity involves the spectrometric analysis of CMC which has been associated with an azo-dye (Remazolbrilliant Blue R)¹¹. In addition, protein activity of both endoglucanases and a range of cellulases can be monitored by the use of a sugar analysis test, such as the p-hydroxy benzoic acid hydrazide (PAHBAH) assay¹². These techniques can be used to elucidate and quantify the activity of the expressed cellulase of interest.

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 and TrCel5A-mCherry Proteins

1. Under sterile conditions, pick single colonies of Agarobacterium tumefaciens (GV3101::pMP90RK GmR, KmR, RifR) containing either pTRAkc-ER-TrCel5A or pTRAkc-ER -TrCel5A-mCherry and transfer to an Erlenmeyer flask containing 10 ml yeast extract broth (YEB, prepared as per manufacturers specifications) medium and kanamycin (50 mg/ml), rifampicin (50 mg/ml), and carbenicillin (100 mg/ml) for selection. Incubate for 36-40 hr at 26 °C and 180 rpm. Note: The design of appropriate constructs for protein expression is outside the scope of this paper, but for guidance, the methods of Maclean et al.¹³ and Munro and Pellham¹⁴ can be followed for construct design.
2. Take 200 µl of each culture to inoculate 20 ml of YEB with the antibiotic concentrations mentioned above and incubate under the same conditions as before.
3. After 36-40 hr, preinduce the cultures by adding 2 µl acetosyringon (200 µM final concentration), 100 µl 40% (w/v) glucose solution and 200 µl of 1 M MES buffer pH 5.6. Incubate overnight.
4. Prepare 100 ml of a 2x infiltration buffer (100 g/L sucrose, 3.6 g/L glucose, 8.6 g/L Murashige and Skoog basal salts, adjust pH to 5.6 using potassium hydroxide).
5. Measure OD₆₀₀ of the cultures with 1:5 dilutions until it reaches an OD₆₀₀ of 5-6. Calculate culture volume needed for 30 ml infiltration medium at OD₆₀₀. Add 15 ml of 2x infiltration buffer, then add deionized H₂O to make the buffer up to 30 ml.
6. Take plants from greenhouse and use a pipette tip to nick the abaxial side of the 3^rd to 5^th leaf from the top to ease infiltration.
7. Use a pipette tip to nick the abaxial side of the leaves to ease infiltration.
8. Fill a syringe with infiltration medium and put it (without needle) on one of the previously made nicks. Support syringe with a finger on the adaxial leaf side. Press the medium carefully into the tissue of interveinal leaf zones. Note: After infiltration, cultivate plants in a greenhouse under the same conditions as described above. Also, maintain an equal proportion of untreated, non-transient expression plants (NTEPs) as controls, under the same conditions as infiltrated plants.

3. Assessment of TrCel5A-mCherry Expression in Plant Leaves

1. After 4-6 days, take plants infiltrated with A. tumefaciens containing pTRAkc-ER-TrCel5A-mCherry from the greenhouse and place them in a dark room for fluorescence analysis.
2. To visualize the fluorescence in leaf sections expressing TrCel5A-mCherry, light plants using a portable light source emitting green light at 515 nm with a red filter (620-750 nm). Note: In depth characterization of protein expression in the plant leaves can be achieved by thin-sectioning leaves with a vibratome and examination under light and fluorescence microscopy. For this follow the steps below:
   1. Cut small sections of approximately 5 x 5 mm size from an infiltrated leaf and embed them in 4% agarose dissolved in 50 mM Phosphate buffer (pH 5.7).
   2. Cut section of 80-90 µm size from embedded leaves using a vibratome. Place them on a microscope slide, cover with buffer or a cover slip and examine them using a microscope at 10X magnification with a fluorescence detector, using the same wavelength and filter stated above.

4. TrCel5A Extraction from Tobacco Leaves

1. Cut pieces from tobacco leaves to an approximate weight of 1 g from plants infected with A. tumefaciens containing pTRAkc-ER-TrCel5A and place in a mortar precooled with liquid nitrogen. Add an appropriate amount of liquid nitrogen to the samples. Grind leaves to powder using a precooled pestle.
2. Add 2 ml of phosphate buffered saline (PBS) containing 1 mM phenylmethylsulfonyl fluoride to the ground leaf matter and mix until the majority of leaf matter is in suspension. Centrifuge extract at 15,000 x g for 20 min at 4 °C and take protein-containing supernatant.
3. Centrifuge the extract at 15,000 x g for 20 min at 4 °C and take protein-containing supernatant being careful not to disrupt the pellet. Discard the pellet.
4. Partially purify TrCel5A by incubating the protein-containing supernatant at 55 °C for 10 min. Allow to rest at room temperature for a further 10 min, then centrifuge at 15,000 x g for 10 min at RT. Repeat partial purification process for non-infiltrated plants to obtain control samples. Use these samples for all following experiments. Note: Protein solutions can be stored at 4 °C for up to 1 week or at -20 °C for up to 3 months.
5. Repeat the partial purification process for non-infiltrated plants to obtain control samples. Use these samples for all following experiments. Note: Protein solutions can be stored at 4 °C for up to 1 week or at -20 °C for up to 3 months.

5. SDS-PAGE, Western Blotting, and Azo-CMC Zymography of TrCel5A-mCherry

1. Purchase or prepare 12% (w/v) SDS-PAGE gels as per established methods¹⁵.
2. Dissolve CMC in water to obtain a 1.5% (w/v) solution. To make CMC-SDS-PAGE gels, substitute 1 ml of 1.5% CMC for 1 ml of water in a 10 ml SDS-PAGE gel mix solution, resulting in a 0.1% (w/v) CMC + 12% (w/v) SDS-PAGE gel mix. Pour gel normally. Note: Make certain that the CMC in the original solution is completely dissolved by a combination of stirring and heating to 40 °C (repeat directly prior to preparing the gel solution).
3. Denature samples by boiling in the presence of SDS, as per established methods¹². As a positive control, use a 1:500 dilution of a commercially available cellulase mixture from T. reesei, such as T. reesei ATCC 26921. As a negative control use a similarly prepared protein solution from non-infiltrated leaves (NTEPs).
4. Carry out the electrophoresis as per normal protocols¹⁵, e.g. use 200 V for ~50 min with the electrophoresis halted once the dye front reached the bottom of the gel.
5. Stain one SDS-PAGE gel using 0.1% (w/v) Coomassie brilliant blue and destain using a methanol/glacial acetic acid mixture. Note: Rapid staining and destaining can be performed by gently heating the staining and destaining solutions, i.e. by using a microwave for ~15 sec to warm (but not boil) first the staining and then the destaining solutions, changing the destaining solution after 10 min, or when cooled, and repeat with new destaining solution.
6. Perform Western blotting of one SDS-PAGE gel using established protocols^16,17. Use the following antibodies: a polyclonal rabbit antibody against a His-tag (primary) and a polyclonal goat anti-rabbit antibody Fc region which has been conjugated to an alkaline phosphatase (secondary). Note: secondary antibodies should be selected to enable visualization using nitroblue tetrazolium chloride/5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (NBT/BCIP), as described¹⁸.
7. Perform in-gel protein refolding on the 0.15% (w/v) CMC SDS-PAGE gel by incubating the gel in 1.5% (w/v) β-cyclodextrin, 20 mM phosphate-citrate buffer, pH 6.5, at room temperature with gentle shaking for 15 min¹⁹.
8. Discard the β-cyclodextrin solution and briefly wash the gel with H₂O. Incubate at RT in 50mM sodium acetate buffer (pH 4.8) for a further 15 min. Perform CMC zymography using the refolded 0.15% CMC SDS-PAGE gel by incubating the gel in 30 mM sodium acetate buffer pH 4.8 for 20 min at 50 °C.
9. Perform CMC zymography using the refolded 0.15% (w/v) CMC SDS-PAGE gel by incubating the gel in 30 mM sodium acetate buffer (pH 4.8) for 20 min at 50 °C.
10. Stain the gel for 10 min using a 0.1% w/v Congo red solution and destain for 30 min or until clear bands are observed using 1 M NaCl. Wash the gel with 0.3% (v/v) acetic acid for clearer imaging.

6. 4-MUC Activity Assay of TrCel5A

1. Prepare a stock solution of 10 mM 4-MUC in 50% (v/v) methanol, and store it in the dark at RT
2. Immediately prior to the assay, prepare a 4-MUC 2x working solution, composed of 1 mM 4-MUC and 60 mM sodium acetate, pH 4.8.
3. Perform this test on samples containing 100 μl of leaf protein (with or without TrCel5A inducibly expressed), 100 μl a commercially available cellulase mixture from T. reesei 1:1,000, and pure H₂O, respectively. Run each sample in triplicate.
4. Pipette 100 μl of the 4-MUC working solution into separate wells of a 96-well plate, then add 100 μl of each sample.
5. Determine 0 min timepoint of 4-MU fluorescence through fluorescent spectroscopy, using an excitation wavelength of 360 nm and an emission wavelength of 465 nm.
6. Incubate plates for 20 min at 50 °C, covered with adhesive lids to prevent evaporation. Stop the reaction by freezing (or by combining 100 μl 0.15 M glycine, pH 10.0, and 100 μl of sample in a separate plate).
7. Determine the endpoint of 4-MU fluorescence through fluorescent spectroscopy, using an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Subtract 0 min from endpoint (20 min) timepoint to obtain the change in fluorescence values.

7. Azo-carboxymethyl Cellulose Activity Assay of TrCel5A

1. Prepare a stock of 1.5% (w/v) Azo-CMC which is stored at RT, and a precipitation solution containing 18 mM zinc acetate, 0.5 M sodium acetate, pH 5 and 80% ethanol (v/v), store at room temperature.
2. Immediately prior to the assay, prepare an Azo-CMC 2x working solution, composed of 1% (w/v) Azo-CMC (in H₂O) and 60 mM sodium acetate, pH 4.8.
3. Combine 50 μl of sample and 50 μl of the working solution in 1.5 ml tubes.
4. Test samples containing 50 μl of leaf protein (with or without TrCel5A transiently expressed); 100 μl of a commercially available cellulase mixture from T. reesei diluted 1:1,000; and pure H₂O. Run samples and standards in triplicate.
5. Incubate samples for 20 min at 50 °C. Stop the reaction by adding 250 μl of precipitation solution. Vortex each sample vigorously for 10 sec to ensure solutions were completely combined.
6. Incubate samples at RT for 10 min and then vortex again. Centrifuge samples at 1,000 x g for 10 min. Remove 200 μl of each sample supernatant to a 96-well plate, taking care not to disrupt the pellet.
7. Assay using absorbance spectroscopy at a wavelength of 590 nm.

8. PAHBAH Assay of TrCel5A

1. Use a hole-punch to cut a circle (~5.5 mm diameter) of filter paper. Add 100 μl 60 mM NaAc, pH 4.8, and 100 μl of samples, either containing leaf protein (with or without TrCel5A inducibly expressed) or a commercial T. reesei cellulase mixture, diluted 1:1,000, and incubate with filter paper circles for 20 hr at 50 °C with 300 rpm shaking. For each sample incubated with filter paper, also incubate a duplicate solution without filter paper as individual sample-controls.
2. Prepare a 330 mM p-hydroxy benzoic acid hydrazide (PAHBAH) solution A by dissolving PAHBAH in H₂O with the addition of 5 ml concentrated HCl for a total of 100 ml solution, and store at RT. Note: Best results are achieved by adding the HCl to a smaller volume of PAHBAH slurry, i.e. 30 ml, which is stirred under temperature to help dissolution before making the volume to 100 ml with H₂O.
3. Prepare PAHBAH solution B, containing 50 mM sodium citrate, 10 mM calcium chloride, and 500 mM NaOH and store at RT.
4. Immediately prior to the assay, prepare a 1.5x working solution of 1 ml PAHBAH reagent to 9 ml buffer solution, store on ice until use (to be used within 4 hr).
5. Prepare samples in thermostable 0.2 or 0.5 ml tubes. Combine 5 μl of each solution incubated with filter paper (step 8.1) 45 μl of H₂O and 100 μl of PAHBAH working solution. Sample controls (samples incubated without filter paper) should be run under the same concentrations (5 μl sample, 45 μl H₂O, 100 μl PAHBAH working solution). Also test 5 standards (5 μl) containing between 0 and 0.2 g/L of glucose, and pure H₂O. Run samples and standards in triplicate.
6. Incubate samples for exactly 10 min at 100 °C, then cool at RT for exactly 10 min. Pipette solutions into a 96 well plate and assay spectroscopically at a wavelength of 410 nm. Subtract individual sample-controls (incubated without filter paper) from samples to obtain final values.

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