Abstract:
Described herein are methods useful for producing proteins, such as enzymes, by agrofiltration. The methods involve producing an  Agrobacterium  with a Ti plasmid encoding a cellulase, infecting plant cells with the  Agrobacterium,  allowing expression of the cellulase, and recovering the cellulase from the plant cells. In one embodiment, the protein produced is an endoglucanase.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. Provisional Application No. 61/090,221, filed Aug. 19, 2008, which is hereby incorporated by reference, in its entirety. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
       [0002]    The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grant No. 0653984 awarded by the National Science Foundation. 
     
    
     BACKGROUND OF THE INVENTION 
       [0003]    Biofuels such as ethanol are fermented from glucose, and the cellulose in biomass is a potential source of this sugar. However, a synergistic set of enzymes is needed to degrade the cellulose into glucose. Typically, these enzymes are produced by fungal cell culture which requires a high capital cost and a large number of bioreactors. Thus, there is a need for a more efficient system of enzyme production that requires lower capital costs, expends less energy, and emits less carbon dioxide. 
       BRIEF SUMMARY OF THE INVENTION 
       [0004]    The present disclosure provides methods of producing a protein, such as a cellulase, by agroinfiltration. The method generally comprises first producing an  Agrobacterium  that contains a modified Ti plasmid encoding the cellulase. The  Agrobacterium  is combined with a plurality of plant cells to form a mixture and to allow infection of at least one plant cell of the plurality. A cellulase-containing fraction is recovered from the mixture after a period of time sufficient for the plant cells to express the cellulase. In one embodiment, the plurality of plant cells is within an intact plant. In another embodiment, the plurality of plant cells is within a detached plant part. In another embodiment, the period of time is at least 4 days. In one embodiment, at least 1 mg cellulase per kg fresh plant cell weight is expressed after the period of time is at least 4 days. In another embodiment, at least 2.6 mg cellulase per kg fresh plant cell weight is expressed after the period of time is at least 6 days. In one embodiment, the cellulase is from a thermophilic organism. In another embodiment, the cellulase is an exoglucanase. In another embodiment, the cellulase is an endoglucanase. In one embodiment, the endoglucanase is β-1,4-endoglucanase E1 from  Acidothermus cellulolyticus.  In one embodiment, the cellulase has an activity of at least 40,000 nmol MU/min/kg fresh plant tissue weight at a pH of 5.5 and a temperature of 65° C. In one embodiment, the  Agrobacterium  is  A. tumefaciens.  In one embodiment, recovering the cellulase-containing fraction comprises rupturing the plurality of plant cells. In another embodiment, expression of the cellulase is under the control of a constitutive promoter. In one embodiment, the constitutive promoter is 35S from cauliflower mosaic virus. In one embodiment, combining the  Agrobacterium  with a plurality of plant cells comprises pressure infiltration. In another embodiment, combining the  Agrobacterium  with a plurality of plant cells comprises vacuum infiltration. In one embodiment, the cellulase is thermostable. In one embodiment, the plurality of plant cells are from  Nicotiana benthamiana.  In one embodiment, the cellulase is linked to a signal peptide. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0005]      FIG. 1  depicts a schematic of the gene synthesized by DNA 2.0, Inc. RAMY 3D SP encodes a signal peptide from rice alpha amylase. E1 is β-1,4-endoglucanase E1 from  Acidothermus cellulolyticus.  E1-cd encodes the E1 catalytic domain. E1-link encodes the E1 linker domain. E1-cbd encodes the E1 cellulose binding domain. PFT-6His encodes a peptide fusion tag, a 6 polyhistidine tag. Stop codons and restriction enzyme sites (XhoI, PstI, HindIII, and SpeI) have been added to flanking regions. 
           [0006]      FIG. 2  depicts the expected amino acid sequence from the β-1,4-endoglucanase translation product. 
           [0007]      FIG. 3  depicts a map of the pDP0701 vector. 
           [0008]      FIG. 4  depicts a map of the pDP07.0202a binary vector. 
           [0009]      FIG. 5  depicts the amount of endoglucanase produced in various tissue samples from tobacco plants ( Nicotiana benthamiana ). Controls #1 and #2 are two different tobacco plants infiltrated with buffer but no bacteria. Experimental #1 and #2 are two different tobacco plants infiltrated with  Agrobacteria  suspended in buffer. Variability was examined between different areas of the same leaf and between leaves of experimental plant #1. Plant to plant variability was examined between experimental plants #1 and #2. 
           [0010]      FIG. 6  depicts transient expression of endoglucanase in tobacco plants. The amount of endoglucanase was monitored over time in infiltrated intact plants and harvested leaves stored in different environments. Hot refers to maximum temperatures &gt;30° C. Cool refers to maximum temperatures &lt;30° C. Light refers to a 16 h/8 h light/dark cycle. Dark refers to 24 h darkness. 
           [0011]      FIG. 7  depicts the modified gene for endoglucanase from  A. cellulolyticus.  The 35S promoter from Cauliflower Mosaic Virus facilitates constitutive transcription. 
           [0012]      FIG. 8  depicts an  Agrobacterium  transferring a specific segment of its Ti plasmid into a plant cell. 
           [0013]      FIG. 9  depicts endoglucanase hydrolyzing β-1,4-glucosidic bonds within cellulose chains (arrows). 
           [0014]      FIG. 10  depicts the optimal conditions for endoglucanase activity. 
           [0015]      FIG. 11  depicts how vacuum infiltration brings  Agrobacteria  and plant cells together. The leaf tissue is immersed in a suspension of  Agrobacteria,  and a vacuum is pulled within the chamber. Air bubbles emerge from the leaf tissue and rise to the surface. The vacuum is released, and the liquid containing the  Agrobacteria  floods the tissue, bringing the bacteria in direct contact with the plant cells. 
           [0016]      FIG. 12  depicts the lab-scale vacuum chamber used to infiltrate intact plants (left) or detached leaves (right). 
           [0017]      FIG. 13  depicts detached leaves 4 days (left), 6 days (middle), and 9 days (right) after infiltration with  Agrobacteria.    
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0018]    The following description sets forth numerous exemplary configurations, parameters, and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present invention, but is instead provided as a description of exemplary embodiments. 
       1. Selection of Initial Target Enzyme and Secretion Signal Peptides 
       [0019]      Acidothermus cellulolyticus  is a thermophilic bacterium that lives in acidic environments. The β-1,4-endoglucanase E1 enzyme was selected from this organism because its ability to hydrolyze cellulose is inhibited at ambient temperatures, so in planta expression of this gene does not alter the plant&#39;s phenotype. Also, the endoglucanase has an optimal activity at pH 5.5, which is approximately the pH of the plant cell apoplast. Furthermore, several other research groups have successfully expressed this enzyme in stable transgenic plants. The sequence for E1 was obtained from the NIH Entrez cross-database search (accession number P54583). The mature protein (without the native secretion signal peptide) consists of 521 amino acids with an estimated molecular weight of 56,477 Da. The protein consists of a catalytic domain (E1-cd, ˜40.3 kDa) and a cellulose-binding domain (E1-cbd, ˜10.8 kDa), connected by a linker region (E1-link, ˜5.4 kDa). The 41-amino-acid native signal peptide was replaced by the 25-amino-acid signal peptide from  Oryza sativa  α-amylase (Ramy3D SP) to facilitate secretion of the protein from plant cells to the apoplast. 
       2. Codon Optimization, Gene Synthesis 
       [0020]    The gene for  A. cellulolyticus  β-1,4-endoglucanase E1 was codon-optimized for expression in  N. benthamiana  using the codon usage table for this plant from the KEGG database. A polyhistidine tag was added to the C-terminus of the protein to allow rapid purification by metal affinity chromatography. Appropriate restriction enzyme sites were added to allow insertion into our other expression cassettes. The entire 1,566 bp DNA fragment was chemically synthesized by an outside company (DNA 2.0, Inc., Menlo Park, Calif.) ( FIG. 1 ). 
         [0000]    3. Cloning into Binary Expression Vectors 
         [0021]    The chemically synthesized E1 gene that encodes β-1,4-endoglucanase from  Acidothermus cellulolyticus  was provided by DNA 2.0 in the vector pJ210: 11772. The coding region of 552aa protein shown in  FIG. 2  contains the 25aa Ramy3D signal peptide fused to the N-terminal and a 6aa his-tag at the C-terminal. 
         [0000]    4. Cloning into the 35S Expression Vector (for Constitutive Expression) 
         [0022]    The vector pJ210: 11772 containing E1 was digested with the restriction endonucleases XhoI and HindIII at positions 1198 and 2872 respectively yielding a 1674 bp fragment that was directionally cloned into the shuttle vector pDE00.0113 creating the plasmid pDP0701. The E1 coding region was cloned downstream from a 35S promoter and upstream from an ocs3′ regulatory sequence creating an E1 35S expression cassette. The E1 expression cassette in pDP0701 ( FIG. 3 ) was excised by digestion with the endonuclease AscI and inserted into the binary vector pDU97.1005 creating the vector designated pDP07.0202a ( FIG. 4 ). 
       5. Creation of Recombinant  Agrobacterium  Strains Containing the 35S Expression Cassette 
       [0023]    The binary plasmid pDP07.0202a was electroporated into the following two  Agrobacterium  strains, EHA105pCH32 and C58C1, resulting in two recombinant  Agrobacterium  ( Agrobacterium tumefaciens ) strains that can be used to transiently express the E1 protein in plant systems. 
         [0000]    6. Production of Recombinant Cellulase Enzyme Using Transient Agroinfiltration in  N. benthamiana    
         [0024]    In the transient expression studies the recombinant EHA105pCH32 Agrobacterial strain with the constitutive CaMV 35S promoter was used. In this expression system, the El transcript is produced under the control of the strong 35S constitutive promoter. This strain of bacteria was cultured in the lab and used to infect four-week-old tobacco ( N. benthamiana ) plants. Infection could take place in the presence or absence of a gene silencing suppressor. The leaves of a 4 week old  Nicotiana benthamiana  plant were vacuum infiltrated. After four days, plant tissue was harvested, homogenized, extracted and tested for enzyme activity. Results are summarized in  FIG. 5 . The minimum amount of enzyme expressed after 4 days was approximately 1 mg cellulase per kg fresh plant cell weight. The activity corresponding to the amounts of enzyme shown in  FIG. 5  ranged from 40,000 to 52,000 nmol MU/min/kg fresh plant tissue weight at pH 5.5 and at 65° C. 
         [0025]    This experiment demonstrated a proof of principle that  A. tumefaciens  can be used to transiently (and rapidly) produce functional endoglucanase in plant tissue. Variability was observed between different tissues and different plants, but in general the yield was 1 mg of enzyme/kg fresh plant weight. Similar results were seen with this constitutive promoter for production of a different protein (human AAT) using this method (Sudarshana et al.  Plant Biotech J.  4: 551-559 (2006)). However, when a viral amplified expression system was used to express AAT, a 70-fold yield increase was achieved, so it is expected that substantial improvements in productivity may be seen when a viral amplicon expression system is used. Also, the activity assay was used to show that the  A. tumefaciens  itself does not produce the enzyme, the plant tissue does. It was also demonstrated that his-tagged rE1 at the C terminal does not eliminate activity. Accordingly, one embodiment of this invention is the functional production of rEl via transient agroinfiltration in plant tissues. 
         [0026]    It was also demonstrated that functional recombinant E1 can be produced in harvested  N. benthamiana  leaves, at even slightly higher expression levels ( FIG. 6 ). In these transient expression studies, the EHA105pCH32 agrobacterial strain was used with the constitutive CaMV 35S promoter. This strain of bacteria was cultured in the lab and used to infect four-week-old tobacco ( N. benthamiana ) plants. The leaves of a 4 week old  Nicotiana benthamiana  plant were vacuum infiltrated. After four days, plant tissue was harvested, homogenized, extracted and tested for enzyme activity. The infiltrated plants and leaves were stored at various conditions to determine their effect on enzyme yield. Intact plants were stored in a hot greenhouse (daily high temperatures &gt;30° C., 14 hours of light per day). To keep the harvested leaves alive, they were stored in a humid container at a constant temperature of 22° C. and protected from light. To make a valid comparison between the plants and leaves, some of each were stored adjacent to each other indoors, at ˜25° C. with 16 hours of light per day. The leaves were stored in a humid container with a clear covering to allow illumination. Intact plants and harvested leaves were tested for enzyme activity after four and six days of incubation. The average amount of enzyme expressed after 6 days was approximately 2.6 mg cellulase per kg fresh plant cell weight. Activity assay results were converted to expression level (mg E1/kg fresh weight plant tissue) based on the reported specific activity of native E1. 
         [0027]    In a further embodiment, activation of the cellulase in planta allows for in situ degradation of cellulose within the leaf tissue. 
         [0028]    Although this E1 embodiment involves the specific example of transient agroinfiltration of rE1 in  N. benthamiana  using a constitutive expression system (CaMV 35S promoter), the approach can be used for production of any cellulose degrading enzyme, including, without limitation, other endoglucanases, exoglucanases, beta-glucosidases, and xylanases, multiple enzymes in the same host plant using co-infiltration, different host plants, and different promoters, plasmids, and expression systems.