# Patent

## Title
Superactive cellulase formulation using cellobiohydrolase-1 from Penicillium funiculosum

## Abstract

Purified cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7A)) enzymes from Penicillium funiculosum demonstrate a high level of specific performance in comparison to other Cel7 family member enzymes when formulated with purified EIcd endoglucanase from A. cellulolyticus and tested on pretreated corn stover. This result is true of the purified native enzyme, as well as recombinantly expressed enzyme, for example, that enzyme expressed in a non-native Aspergillus host. In a specific example, the specific performance of the formulation using purified recombinant Cel7A from Penicillium funiculosum expressed in A. awamori is increased by more than 200% when compared to a formulation using purified Cel7A from Trichoderma reesei.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 10/557,589, filed Nov. 17, 2005, which is a national stage application of International Application No. PCT/US03/06172, filed Feb. 27, 2003. The entire disclosure of U.S. application Ser. No. 10/557,589 and International Application No. PCT/US03/06172 is incorporated herein by reference.


CONTRACTUAL ORIGIN OF THE INVENTION
The United States Government has rights in this invention under Contract No. DE-AC36-08GO28308 between the United States Department of Energy and the Alliance for Sustainable Energy, LLC, Manager and Operator of the National Renewable Energy Laboratory.


TECHNICAL FIELD
The invention pertains to the field of cellulases and, particularly, members of the glycosyl hydrolase Cel7 family, which is also known as the cellobiohydrolase I (CBH I) family of enzymes. More particularly, novel formulations using either purified native P. funiclosum Cel7A or purified recombinant P. funiculosum Cel7A (rCel7A) expressed from a transgenic Aspergillus host exhibit superior performance on pretreated corn stover compared with that formulation using purified Trichoderma reesei Cel7A.
BACKGROUND ART
Cellulosic biomass is a favorable feedstock for fuel ethanol production because it is both readily available and less expensive than either corn or sugarcane. Nevertheless, substantial problems must be overcome before a typical cellulosic feedstock can be utilized effectively and economically as a substrate for the fermentative production of ethanol. By way of example, cellulosic biomass feedstocks may include wood pulp or agricultural residues, such as corn stover, straw, grass, or weeds. A typical feedstock is comprised of approximately 35-45% cellulose, 30-40% hemicellulose, 15% lignin and 10% of other components. The cellulose fraction is composed of linear (and to a substantial extent, microcrystalline) polymers of the hexose sugar, glucose. Saccharification of cellulose releases sugars, which may be converted into ethanol or other products by fermentation. The hemicellulose fraction is comprised mostly of pentose sugars, including xylose and arabinose.
Alcohol products derived from cellulosic biomass are relatively expensive when compared to analogous fuels from other sources. A significant cost factor is the need to provide hydrolyzing enzymes, such as cellulases, that attack the cellulosic and/or hemicellulosic substrates to release sugars. These enzymes are produced by microorganisms, and may be purified from fermentation broth. The cost of cellulase is presently a significant component of the overall cost of biomass-derived ethanol. In the United States, ethanol production is heavily subsidized by tax incentives that encourage the use of ethanol in reformulated gasoline.
A variety of cellulases are known. Table 1 below lists various cellulases of the Cel7 family. Cel7 enzymes are the principal component in commercial cellulase formulations—typically accounting for most of the actual bond cleavage in the saccharification of cellulose. Cel7 cellobiohydrolases are members of the Class of beta proteins, the Superfamily of concanavalin A-like lectins/glucanases, and the Family of glycosyl hydrolase family 7 catalytic core proteins. The Cel7 family of enzymes may differ from one another by various insertions, deletions, and alterations in the catalytic domain and linker peptide. The cellulose binding domain of Cel7 enzymes is highly conserved. Cel7A from Trichoderma reesei is the most widely used CBH I commercial enzyme because it is capable of withstanding commercial process conditions and demonstrates the highest known level of saccharification in the entire family.







TABLE 1







VARIOUS CELLULOSE 1,4-β-CELLOBIOSIDASE MEMBERS












GenBank/GenPept



Enzyme
Organism
Accessions
Swiss Prot





Cellobiohydrolase I
Agaricus
Z50094 CAA90422.1
Q92400



bisporus


exoglucanase C1
Alternaria
AF176571 AAF05699.1



alternata


Cellobiohydrolase I
Aspergillus
AB002821 BAA25183.1
O59843



aculeatus


Cellobiohydrolase
Aspergillus
AF420019 AAM54069.1


(CbhA)
nidulans


Cellobiohydrolase
Aspergillus
AF420020 AAM54070.1


(CbhB)
nidulans


Cellobiohydrolase A
Aspergillus niger
AF156268 AAF04491.1


(CbhA)


Cellobiohydrolase B
Aspergillus niger
AF156269 AAF04492.1
Q9UVS8


(CbhB)


Cellobiohydrolase I
Claviceps
Y07550 CAA68840.1
O00082



purpurea


Cellobiohydrolase I
Cochliobolus
U25129 AAC49089.1
Q00328



carbonum


Cellobiohydrolase I
Cryphonectria
L43048 AAB00479.1
Q00548



parasitica


Cellobiohydrolase I
Fusarium
L29379 AAA65587.1
P46238


(Cel7A)
oxysporum


Cellobiohydrolase 1.2
Humicola grisea
U50594 AAD11942.1
O94093




AAN19007.1


Cellobiohydrolase 1
Humicola grisea
D63515 BAA09785.1
P15828




X17258 CAA35159.1
Q12621


Cellobiohydrolase I
Humicola grisea
AB003105 BAA74517.1
O93780



var. thermoidea


Cellobiohydrolase I.2
Humicola grisea
AF123441 AAD31545.1



var. thermoidea


Cellobiohydrolase I
Melanocarpus



albomyces


Cellulose 1,4-β-
Melanocarpus
AJ515705 CAD56667.1


cellobiosidase
albomyces


(Cel7B)


Cellobiohydrolase I
Neurospora
X77778 CAA54815.1
P38676



crassa


Cellobiohydrolase
Penicillium
AJ312295 CAC85737.1



funiculosum


Cellobiohydrolase I
Penicillium
S56178 CAA41780.1
Q06886



janthinellum
X59054 CAA41780.1


Cellobiohydrolase
Phanerochaete
S40817 AAA09708.1
Q01762



chrysosporium
X54411 CAA38274.1


Cellobiohydrolase I-1
Phanerochaete
M22220 AAB46373.1
P13860



chrysosporium
Z22528 CAA80253.1


Cellobiohydrolase I-2
Phanerochaete
L22656 AAA19802.1
Q09431


(Cel7D)
chrysosporium
Z11726 CAA77789.1




Z11733 CAA77795.1




Z22527 CAA80252.1




Z29653 CAA82761.1




Z29653 CAA82762.1


Cellobiohydrolase 1
Talaromyces
AF439935 AAL33603.2


(Cbh1A)
emersonii
AY081766 AAL89553.1


Cellobiohydrolase I
Trichoderma
X69976 CAA49596.1
P00725


(Cel7A)
reesei



(Hypocrea



jecorina)


Cellobiohydrolase I
Trichoderma
X53931 CAA37878.1
P19355



viride


Cellobiohydrolase I
Trichoderma
AB021656 BAA36215.1
O93832



viride


Cellobiohydrolase I
Volvariella
AF156693 AAD41096.1


(CbhI)
volvacea V14









Cellulases often demonstrate enzymatic synergy in mixtures with other hydrolyzing enzymes; for example, between one enzyme that attacks cellulose and another that attacks hemicellulose. Various efforts have been made to provide transgenic organisms with one or more recombinant genes and obtain multiple functionality from a single organism, for example, as described in U.S. Pat. No. 5,536,655 issued to Thomas et al. for the gene encoding EI endoglucanase from Acidothermus cellulolyticus. 
United States patent application publication US 2002/0155536 to Van en Brink et al. discloses a method of isolating DNA sequences coding for one or more proteins of interest advantageously using an Aspergillus host. More specifically, cDNA is prepared from an organism of interest. Fragments of the cDNA are inserted into a vector to obtain a cDNA library. Subsequent transformation of the cDNA library into filamentous fungi, such as Aspergillus, facilitates screening for clones that express proteins of interest.
The '536 patent publication describes an expression system using filamentous fungi, such as Aspergillus, to provide host cells to screen for proteins of interest. Expression in an Aspergillus host renders the cloned polypeptide sequences more easily detectable due to a higher secretory capacity and less glycosylation, as by way of example, in Aspergillus niger as compared to yeast. The '536 patent does not teach that P. funiculosum Cel7A can be secreted from Aspergillus awamori with full functionality.
United States patent application publication US 2002/0061560 to Lawlis describes a method of obtaining a secretory protein at a higher level in filamentous fungi, for example, Aspergillus awamori. More specifically, the coding sequence for the protein of interest is fused with DNA fragments encoding signal peptide, a cleavable linker peptide, and a portion of a protein native to the filamentous fungal host (i.e., protein that is normally secreted from Aspergillus). The '560 publication pertains to increased quantities of secreted proteins, and does not teach that P. funiculosum Cel7A can be secreted with full functionality.
WO 92/06209 to Ward et al. relates to an improved process for transforming the filamentous fungus T. reesei. T. reesei cells are treated with homologous DNA originally derived from T. reesei. The homologous DNA is provided with a selectable marker, which is used to select transformants. Although CBH I is used as an example, nothing related specifically to the processing and secretion of P. funiculosum Cel7A is taught or disclosed.
Efforts in recombinant technologies that pertain to the production of cellulases emphasize the production of cellulase in greater quantity or the production of cellulase having greater activity measured as conversion efficiency over time. It is notoriously difficult to compare the activity or performance of cellulases on naturally occurring cellulosic substrates. The naturally occurring substrates vary in composition, which makes it difficult to provide a uniform basis of comparison. Additionally, one is prone to draw unwarranted conclusions where higher concentrations of enzymes may produce surface effects when the enzyme interferes with itself. Similarly unwarranted conclusions may be drawn where adsorption effects (i.e., enzyme loss) impair the activity of lower cellulase concentrations. When it becomes necessary to measure performance with exactitude, commercial enterprises often choose to consult institutions, such as the National Renewable Energy Laboratory located in Golden, Colo.
Transgenic expression of genes does not necessarily result in the production of useful cellulase. For example, glycosylation by yeast used to express the Cel7 family enzymes may render the enzymes less effective or ineffective. The choice of host organism is limited to those organisms that can survive commercial process conditions, for example, the Direct Microbial Conversion (DMC) process or the Simultaneous Saccharification and Fermentation (SSF) process. In the DMC method, a single microbial system both produces cellulase and ethanol as a fermentation product. The SSF method utilizes two biological elements, one that produces cellulase enzyme and the other, which ferments sugar to ethanol. The DMC process is described in (Brooks et. al., Proc. Annu. Fuels Biomass Symp., 2nd (1978). The SSF process is described in Ghose et. al., Biotechnol. Bioeng., (1984), 26 (4): 377-381 (1984)., e.g., as described by Spindler et al, Biotechnology Letters, 14:403-407 (1992). By way of example, SSF process conditions may impose a pH of 4.5 to 5.5 and a temperature from 30° C. to 38° C. It can be difficult to choose a suitable host capable of both expressing a useful form of the cellulase of interest and surviving the process conditions where the cellulase is also active under process conditions.
SUMMARY
The present invention advances the art and overcomes the problems outlined above by providing a cellobiohydrolase I family 7 (Cel7) enzyme formulation using the native Cel7A from Penicillium funiculosum or its recombinant analogue expressed in a transgenic filamentous fungi, e.g., Aspergillus sp. The Cel7A enzyme formulation demonstrates an unusually high (if not unprecedented) level of specific performance in saccharification of cellulose.
The Cel7 enzyme formulation contains Cel7A enzyme obtained from P. funiculosum or a recombinant analog thereof, for example, a polypeptide having a sequence of SEQ ID NO. 9. The Cel7A enzyme may be combined with an endoglucanase present in amount ranging from one percent (1%) to twenty percent (20%) of the molar concentration of the Cel7A enzyme.
The Cel7A enzyme may be a recombinantly expressed enzyme from a transgenic filamentous fungus. Examples of the transgenic filamentous fungus include Aspergillus sp., Trichoderma sp., and Penicillium sp. Further examples of the transgenic filamentous fungus include Aspergillus awamori, Aspergillus niger, Trichoderma reesei, and even Penicillium funiculosum expressing a transgene encoding the Cel7 enzyme under control of a promoter that is non-native to Penicillium funiculosum. 
The cellobiohydrolase formulation exhibits specific performance in saccharification of biomass cellulose that exceeds by at least two-fold (i.e., 200%) the specific performance of a comparable cellobiohydrolase formulation where Cel7A cellulase from Trichoderma reesei is substituted for the Cel7A enzyme. This level of specific performance is heretofore unknown from Cel7 family enzymes. By way of example, the specific performance may be measured on pretreated corn stover.
The endoglucanase may be EI endoglucanase mixed with the Cel7 enzyme, for example, in a 95:5 molar ratio of the Cel7A enzyme to EI endoglucanase. Additional hydrolyzing enzymes, such as β-glucosidase, may be added to the mixture.
Recombinant expression of the Cel7 may be achieved by transforming a filamentous fungus with a gene from Penicillium funiculosum. The gene encodes a Cel7 enzyme that is foreign to the filamentous fungus and/or places the coding region under the control of a non-native promoter. The recombinant Cel7 (rCel7) is expressed by the filamentous fungus using biological processes that operate on the nucleic acid sequence to produce rCel7. The rCel7 enzyme may be purified, e.g., by chromatography, to a concentration that exceeds a concentration obtainable from natural expression processes.
A specific embodiment of the instrumentalities described herein should not be interpreted to define the invention in unduly narrow terms. This is because the specific embodiment teaches by way of example, and not by limitation. Aspergillus awamori is used as a host organism to express a particular form of rCel7A enzyme, namely, using a gene encoding Cel7A isolated from Penicillium and, more particularly, from Penicillium funiculosum. 
The Cel7A enzyme represented by SEQ ID NO. 9, after expression by A. awamori, is characterized by a thermal denaturation temperature of 66.8° C., as measured by differential scanning microcalorimetry (DSC) at pH 5.0 and a scan rate of 60° C./hour.
Standards for measuring specific performance and other characteristics of cellulases are known in the art. For example, useful techniques of this type are taught by “Hydrolysis of Cellulose Using Ternary Mixtures of Purified Cellulases,” J. O. Baker, C. I. Ehrman, W. S. Adney, S. R. Thomas, and M. E. Himmel, Appl. Biochem. Biotechnol., 70/72, 395-403, (1998); and “A Membrane-Reactor Saccharification Assay to Evaluate the Performance of Cellulases Under Simulated SSF Conditions,” J. O. Baker, T. B. Vinzant, C. I. Ehrman, W. S. Adney, and M. E. Himmel, Appl. Biochem. Biotechnol., 63-65, 585-595, (1997).
Different substrates may be used in testing the Cel7 enzyme formulation. For example, pretreated wood pulp or pretreated agricultural residues may be used for comparative testing purposes to confirm the superior specific performance. Pretreated corn stover is often used as a basis of comparison. Efficient enzymatic hydrolysis of these substrates by cellulase often requires the synergistic cooperation of at least two types of enzymes, namely, endoglucanases and exoglucanases, such as cellobiohydrolase. According to these protocols, the specific performance may be measured using a mixture of enzymes, for example, a mixture of rCel7A (or native Cel7A) and endoglucanase at a molar ratio of 95:5.
Increased specific performance of Cel7A may be facilitated in reaction processes by the addition of other hydrolyzing enzymes, such as β-glucosidase. A particularly useful enzyme mixture includes T. reesei Cel7A mixed in a 95:5 molar ratio with A. cellulolyticus EIcd endoglucanase, such as EI endoglucanase expressed from the Acidothermus cellulolyticus gene reported in U.S. Pat. No. 5,536,655 issued to Thomas et al., which is incorporated by reference herein. Another useful additional enzyme is β-glucosidase from Aspergillus niger, as reported in “Hydrolysis of Cellulose Using Ternary Mixtures of Purified Cellulases,” J. O. Baker, C. I. Ehrman, W. S. Adney, S. R. Thomas, and M. E. Himmel, Appl. Biochem. Biotechnol., 70/72, 395-403, (1998). This formulation shows superior performance. Other hydrolyzing enzymes that may be included in such mixtures include members of Family Cel6.
The P. funiculosum rCel7A enzyme may be purified from cultures of native organisms or recombinant hosts. Purification may be accomplished by chromatography to levels of, for example, 50%, 75%, 90%, or research grade purity.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic block diagram that shows a process for producing a recombinant Cel7A enzyme;
FIG. 2 shows DNA and polypeptide sequences of interest for Cel7A enzyme isolated from P. funiculosum 
FIG. 3 depicts a vector that may be recombined with the Cel7 coding region and/or a promoter sequence for use in transforming the filamentous fungus according to the instrumentalities described herein;
FIG. 4 depicts the vector of FIG. 3 after recombination;
FIG. 5 shows a comparison of the specific performance of various Cel7A enzyme formulations (i.e., Cel7A or rCel7A and EIcd endoglucanase) on pretreated corn stover using the DSA assay, where the comparison includes that for standard loadings of purified recombinant Cel7A from P. funiculosum expressed in A. awamori; purified native Cel7A from P. funiculosum; and purified native Cel7A from T. reesei. Also illustrated in FIG. 5 is the one-half loading of purified recombinant Cel7A from P. funiculosum expressed in A. awamori. 
FIG. 6 provides a comparison of thermal transition temperatures between various purified Cel7A enzymes using DSC.



DETAILED DESCRIPTION
There will now be shown and described a process for the production of rCel7 in Aspergillus and, specifically P. funiculosum rCel7A expressed in A. awamori. The presentation of a specific embodiment according to the various embodiments and instrumentalities described herein should not unduly limit the scope of the invention, because the teaching is by way of example.
Procedures for constructing recombinant molecules are disclosed by Sambrook et al., supra. Briefly, a DNA sequence encoding a Cel7A enzyme from Penicillium funiculosum, or its functional derivatives exhibiting Cel7 activity, may be recombined with vector DNA in accordance with conventional techniques. These techniques and materials include, for example, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and blunt-ended or cohesive-ended ligation with appropriate ligases. Part or all of the genes may be synthesized chemically in overlapping fragments which are hybridized in groups and ligated to form longer double-stranded DNA molecules. The resulting vector may then be introduced into a host cell by transformation, transfection, techniques such as electroporation, etc. Techniques for introducing a vector into a host cell are well known.
A vector is a DNA molecule, often derived from a plasmid, bacteriophage or hybrid, into which fragments of DNA may be inserted or cloned. A vector usually contains one or more unique restriction sites, and may be capable of autonomous replication or integration into the genome of a defined host organism such that the cloned sequence is reproducible.
FIG. 1 is a process diagram 100 showing steps for the production of rCel7A from P. funiculosum. In step 102, a culture of a source organism, such as the source organisms identified above in Table 1, are screened for Cel7 activity. For example, P. funiculosum (ATCC62998) is screened to identify the presence of Cel7A. In step 104, primers are constructed based upon the presumed commonality of conserved sequences among the respective domains, such as the catalytic domain in the Cel7A family. The gene is then isolated by polymerase chain reaction (PCR). Step 106 entails inserting the gene into an expression vector, for example, using overlap extension PCR to insert the gene into expression vector pFE2. In step 108, the expression vector is used in host transformation, specifically protoplast transformation of Aspergillus awamori ATCC22342. A transformed strain that is confirmed to carry the rCel7A gene is grown for a period of time in step 110, and the rCel7A is purified in step 112.
The Cel7A family genes can be cloned for expression in Aspergillus and other filamentous fungi using recombinant DNA techniques, as will be described below and illustrated by specific example. Variations on laboratory techniques are well known and may be adapted to implement the instrumentalities described herein. In addition to the disclosed embodiments, the DNA cloning process can be facilitated through a variety of other means, such as application of recombinant DNA techniques, the polymerase chain reaction techniques (PCR) or DNA synthesis of the gene. Techniques for synthesizing oligonucleotides are disclosed by, for example, Wu et al, Prog. Nucl. Acid. Res. Molec. Biol. 21:101-141 (1978).
Standard reference works setting forth the general principles of recombinant DNA technology and cell biology include, for example, Watson et al., Molecular Biology of the Gene, Volumes I and II, Benjamin/Cummings Publishing Co., Inc., Menlo Park, Calif. (1987); Darnell et al., Molecular Cell Biology, Scientific American Books, Inc., New York, N.Y. (1986); Lewin, Genes II, John Wiley & Sons, New York, N.Y. (1985); Old et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2nd Ed., University of California Press, Berkeley, Calif. (1981); Sambrook et al, (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)) and Albers et al., Molecular Biology of the Cell, 2nd Ed., Garland Publishing, Inc., New York, N.Y. (1989).
The following nonlimiting examples establish preferred materials and methods for practicing the process steps of FIG. 1, as well as for use in comparing the specific activities of rCel7A enzymes
EXAMPLE 1
Isolating a Cel7A Gene from P. funiculosum 
The coding sequence for the cel7A gene from P. funiculosum (ATCC 62998) (SEQ ID NO. 7 of FIG. 2) was inserted and expressed in the fungal host Aspergillus awamori (ATCC 22342) using the fungal expression vector pFE2. As shown in FIG. 3, the pFE2 vector is an E. coli-Aspergillus shuttle vector, and contains elements required for maintenance in both hosts. The pFE2 vector directs the expression and secretion of the protein of interest as a fusion protein with a portion of the glucoamylase (GA) gene fused to the cel7A gene. The vector contains the Streptoalloteichus hindustanus phleomycin resistance gene (Phleo) under the control of the A. niger β-tubulin promoter, for positive selection of Aspergillus transformants. The vector also contains a β-lactamase gene for positive selection using ampicillin in E. Coli, and the A. niger trpC terminator (trpC T). In this case, the rCel7 protein from P. funiculosum was expressed with its own secretion signal peptide by replacing the glucoamylase signal by PCR overlap extension and was expressed under the control of the Aspergillus niger GA promoter.
As shown in FIG. 4, the construct used to produce recombinant Cel7 enzyme was designated pFEcbhI(Pf)3726 and was designed to contain the native P. funiculosum signal sequence (SEQ ID. NO. 8; Pf signal) with the native coding sequence (P. funiculosum cbhI gene) for the structural protein. The 1.5-kb P. funiculosum cbhI structural gene (including the signal sequence starting with ATG codon and the mature protein coding sequence, which ends with TAG stop codon) was used to replace the GA signal sequence in the pFE2 vector. The GA signal sequence was replaced using overlap extension PCR where the native P. funiculosum structural gene was placed precisely under the GA promoter and regulatory region in pFE2. In addition, a NotI restriction site was added after the TAG codon (see primer PfcbhINotI(R)) to position the gene immediately before the trpC T terminator region in pFE2.
The regulatory region containing GA promoter and native P. funiculosum Cel7A signal region (0.9 kb) was PCR amplified using PfuI with the following oligonucleotide primers:










(SEQ ID NO. 1)











FE2-2(F):




5′GTATACACGCTGGATCCGAACTCC 3′



 










(SEQ ID NO. 2)











FE2-PfcbhI(R):




5′GTTCAAGGCAGACATTGCTGAGGTGTAATGATGC 3′



[5′PfcbhI signal(tail)] [Sequence before GA



signal]






The template used for the PCR was pFE2 vector. The bolded region of SEQ ID NO. 2 above anneals to the sequence immediately before the GA signal sequence in pFE2. The underlined region is complementary to the 5′ P. funiculosum CBH I signal coding sequence. After this round of PCR, the regulatory region included the GA promoter region from pFE2 and 5′ end of native P. funiculosum Cel7A signal sequence.
The structural gene of P. funiculosum Cel7A was amplified using the following primers:








(SEQ ID NO. 3)









PfcbhI-FE2(F):



5′GCATCATTACACCTCAGCAATGTCTGCCTTGAAC 3′


[Sequence before GA signal (tail)] [5′Pf CBH I


signal]


 








(SEQ ID NO. 4)









PfcbhINotI(R):



5′ATAAGAATGCGGCCGCCTACAAACATTGAGAGTAGTAAGGG 3′.






Genomic DNA of P. funiculosum (ATCC 62998) was used as template for the PCR reaction. The bolded sequence in PfcbhI-FE2 (F) primer (SEQ ID NO. 3) annealed with pFE2 vector and not the P. funiculosum cel7A coding sequence. Following this round of PCR the 5′-end of the cel7A structural gene contained the extra sequence of the GA signal.
To combine the GA promoter and GA regulatory region from pFE2 and the P. funiculosum cel7 structural gene, a third round of PCR was conducted using the two PCR products described above and primers FE2-2 (F) (SEQ ID NO. 1) and PfcbhINotI(R) (SEQ ID NO. 4). The product (2.6 kb) was ligated in a T/A cloning vector, pGEMTeasy, which is obtainable on commercial order from Promega of Madison, Wis., and used to transform E. coli DH5α. Transformants with correct inserts were identified by colony PCR screening using a primer pair including:










P13 (F):
ACTTCGTTACCGGCTCTAACG
(SEQ ID NO. 5)



and


 


Pf4(R):
GAAGTCACATCCGTCAGGGTC.
(SEQ ID NO. 6)






The primer pair Pf3 (F) (SEQ ID NO. 5) and Pf4(R) (SEQ ID NO. 6) is internal to P. funiculosum Cel7A. Plasmid DNA was extracted from several of these correct transformants and digested with BamHI followed by Klenow polymerase treatment to generate the blunt ends. BamHI is located in the 5′ end of the 2.6-kb fragment and intrinsic to the GA promoter region of pFE2 vector. A 2.4-kb fragment was obtained by NotI digestion of the BamHI/Klenow linearized plasmid. NotI was included in the PfcbhINotI(R) for cloning purposes. To transfer this 2.4-kb fragment into pFE2, pFE2 was digested with SacI and blunt-ended with T4 DNA polymerase followed by digestion with NotI. A 5.5-kb vector fragment was recovered by gel extraction and ligated to the 2.4-kb fragment. The recombinant plasmid was named pFcbhI(Pf)3726.
After transformation in DH5V and confirmation of the plasmid by colony PCR and restriction digestion pattern, the plasmid was used to transform A. awamori by spheroplast transformation. Zeocin-resistant transformants were grown in liquid medium CMZ300 (CM+Zeocin 300 ug/mL) for 3-4 days and supernatant analyzed by activity on p-nitrophenol β-D lactopyranoside activity assay. One of the positive clones was grown in 1-L CMZ300 medium for 7-10 days and supernatant subjected to protein purification and analysis. Expression products of this clone were confirmed to contain the recombinant Cel7A enzyme isolated from P. funiculosum (SEQ ID NO. 9).
EXAMPLE 2
Transforming A. awamori 
Aspergillus awamori (ATCC22342) spore stocks were stored at −70° C. in 20% glycerol, 10% lactose. After thawing, 200 μL of spores were inoculated into 50 mL CM-glucose broth (5 g·L−1 Yeast Extract; 5 g·L−1 Tryptone; 10 g·L−1 Glucose; 20× Clutterbuck's Salts (120.0 g·L−1 Na2NO3; 10.4 g·L−1 KCl; 10.4 g·L−1 MgSO4.7H2O; 30.4 g·L−1 KH2PO4) 50 mL·L−1) at pH 7.5 in each of eight-baffled 250 mL Erlenmeyer flask. The cultures were grown at 28° C., 225 rpm for 48 h.
The mycelial balls were removed by filtration through sterile Miracloth (Calbiochem, San Diego, Calif.) and washed thoroughly with sterile KCM (0.7M KCl, 10 mM MOPS, pH 5.8) to remove ungerminated spores. Approximately 10 g wet weight of washed mycelia were transferred to 50 mL KCM+250 mg Lysing Enzyme from Trichoderma harzianum (Sigma-Aldrich, St. Louis, Mo.) in a 250 mL baffled Erlenmeyer flask. The digestion mixture was incubated overnight at 30° C., 80 rpm.
Following digestion, the mycelia were titurated with a 25 mL disposable pipette to loosen the hyphal cells and filtered through sterile Miracloth into 50 mL conical centrifuge tubes. The spheroplasts were pelleted at 1500×g for 12 min and resuspended in 0.7M KCl by gentle tituration with a 25 mL pipette. This was repeated once. After a third pelleting, the spheroplasts were resuspended in 10 mL KC (0.7M KCl; 50 mM CaCl2), pelleted and resuspended in 1.0 mL KC using a wide-bore pipet tip.
The washed spheroplasts were transformed by adding 12.5 μL PCM (40% PEG 8000; 50 mM CaCl2; 10 mM MOPS pH 5.8) and 5 μL DNA ( ̃0.5 μg/μL) to 50 μL of spheroplasts in sterile 1.5 mL Eppendorf tubes. After incubation on ice for 45 minutes, 0.5 mL of room temperature PCM was added to the transformation mixture and was mixed by inversion and gentle vortexing. The mixture was incubated at room temperature for 45 minutes. One milliliter of KC was added and mixed. Selection of transformants was by zeocin resistance. The mixture was allocated between four tubes (10 mL each) of CM top agar at 55° C., which were each poured over a 15 mL CM plate with 170 μg/mL zeocin. The plates were incubated at 28° C. for 2-3 days. Subsurface colonies were partially picked with a sterile wide bore pipet tip, exposing the remaining part of the colony to air and promoting rapid sporulation. After sporulation, spores were streaked onto several successive CM plates with either 100 or 300 μg/mL zeocin added. After a monoculture was established, heavily sporulated plates were flooded with sterile spore suspension medium (20% glycerol, 10% lactose), the spores were suspended and aliquots were frozen at −70° C. Working spore stocks were stored on CM slants in screw cap tubes at 4° C. The recombination event was through random integration of the plasmid into the A. awamori genome.
EXAMPLE 3
Culturing A. awamori 
For enzyme production, spores were inoculated into 50 mL CM basal fermentation medium (5.0 g·L−1 Enzymatic Casein Hydrolysate; 5.0 g·L−1 NH4CL; 10.0 g·L−1 Yeast Extract; 10.0 g·L−1 Tryptone; 2.0 g·L−1 MgSO4.7H2O; 50.0 g·L−1 Soluble Starch; 50 mM Bis-Tris-Propane, pH 7.0), and grown at 32° C., 225 rpm in 250 mL baffled flasks. The cultures were transferred to 1.0 L of basal fermentation medium in 2800 mL Fernbach flasks and grown under similar conditions. The flasks were harvested by filtration through Miracloth after 7-10 days of growth.
EXAMPLE 4
Purifying rCel7A from A. awamori Culture Broth
The purification of rCBH 1 from A. awamori fungal broths was started by filtration through glass fiber filters followed by concentration of the broth by using a 500 mL Amicon stirred cell concentrator with PM-10 cutoff filters. After the broth was concentrated to a volume of approximately 50 mL and was then extensively diafiltered by successive dilution and concentration with the stirred cell with 50 mM Bis-Tris pH 5.8 buffer to a point at which the conductivity of the solution was less than 2 mS/cm. The sample was then applied to a HiPrep 16/10 DEAE FF column (Amersham Biosciences) equilibrated with 50 mM Bis-Tris, pH 5.8 buffer with a flow rate of 10 mL/min at 4° C. After the sample was loaded and the column washed extensively with equilibration buffer the bound fraction was eluted with a linear gradient of 0 to 1.0 M NaCl in the same equilibration buffer. The fractions containing activity on p-nitrophenol β-D-lactopyranoside were pooled and concentrated to a final volume of 10 mL using Amicon stirred cell concentrators and PM-10 cutoff filters (10,000 kDa nominal molecular weight cutoff). The enzyme was further purified and the buffer exchanged by means of size exclusion chromatography using a HiLoad 26/60 Superdex 200 column (Amersham Biosciences) in 20 mM acetate, 100 mM NaCl, pH 5.0 buffer. At this point the protein eluted as a single, symmetrical peak and the purity was confirmed as a single band when analyzed with a NuPage 4-12% Bis-Tris gradient gel using MOPS-SDS buffer (Invitrogen) according to the manufactures recommended conditions. Concentrations of purified proteins were determined by absorbance at 280 nm using the extinction coefficient and molecular weight calculated for Cel7A by the ProtParam Software obtained from the ExPASy website.
EXAMPLE 5
Production and Purification of Native Penicillium funiculosum Cel7A
Native Cel7A protein was produced from Penicillium funiculosum grown in two 500 mL cultures in CM-PSC broth (5 g·L−1 Yeast Extract; 10 g·L−1 phosphoric acid swollen cellulose; 5 g·L−1 Tryptone; 20× Clutterbuck's Salts (120.0 g·L−1 Na2NO3; 10.4 g·L−1 KCl; 10.4 g·L−1 MgSO4.7H2O; 30.4 g·L−1 KH2PO4) 50 mL·L−1) at pH 7.5 in baffled 1 L Erlenmeyer flasks. The flasks were inoculated with 1 mL of a spore suspension of P. funiculosum ATCC 62998 and incubated at 28° C. in a New Brunswick Scientific Model Innova 4230 shaking incubator at 225 rpm. The cultures were harvested by filtration following 7 days of growth.
The purification of the native Cel7 from P. funiculosum broth was started by filtration through glass fiber filters followed by concentration of the broth by using a 500 mL Amicon stirred cell concentrator with PM-10 cutoff membranes. The broth was first concentrated to a volume of approximately 50 mL followed by extensive diafiltration by successively diluting and concentrating the broth with 50 mM Bis-Tris pH 5.8 buffer to a point at which the conductivity of the solution was less than 2 mS/cm. The sample was then applied to a 6 mL Resource Q column (Amersham Biosciences) equilibrated with 50 mM Bis-Tris, pH 5.8 buffer with a flow rate of 6 mL/min. After the sample was loaded and the column washed extensively with equilibration buffer and the bound fraction was eluted with a linear gradient of 0 to 1.0 M NaCl in the same equilibration buffer. The fractions containing activity on p-nitrophenol β-D lactopyranoside were pooled and concentrated to a final volume of 10 mL using Amicon stirred cell concentrators and PM-10 cutoff filters (10,000 kDa nominal molecular weight cutoff). The enzyme was further purified and the buffer exchanged by means, of size exclusion chromatography using a HiLoad 26/60 Superdex 200 column (Amersham Biosciences) in 20 mM acetate, 100 mM; NaCl, pH 5.0 buffer. The purity was confirmed as a single band using a NuPage 4-12% Bis-Tris gradient gel and MOPS-SDS buffer (Invitrogen) according to the manufacturer's recommended conditions. The concentration of the purified native Cel7 solution was determined by absorbance at 280 nm using the extinction coefficient and molecular weight calculated for Cel7A by the ProtParam tool on the ExPASy website.
EXAMPLE 6
Comparative Testing of Specific Performance
The purified rCel7A and Cel7A from Examples 4 and 5 were tested against other Cel7A enzymes. Test procedures included those described in “Hydrolysis of Cellulose Using Ternary Mixtures of Purified Cellulases,” J. O. Baker, C. I. Ehrman, W. S. Adney, S. R. Thomas, and M. E. Himmel, Appl. Biochem. Biotechnol, 70/72, 395-403, (1998), and “A Membrane-Reactor Saccharification Assay to Evaluate the Performance of Cellulases Under Simulated SSF Conditions,” J. O. Baker, T. B. Vinzant, C. I. Ehrman, W. S. Adney, and M. E. Himmel, Appl. Biochem. Biotechnol, 63-65, 585-595, (1997).
Diafiltration saccharification assays (DSA) were performed as disclosed in the above article by Baker et al. (1997) with the modifications that the membrane installed in the cells was a BioMax-5 (5,000 Da nom. MWCO, Millipore Corp.) rather than a PM-10 (10,000 Da nom. MWCO, Amicon, Inc.), and the buffer flow rate through the membrane was 0.020 mL/min. All assays were at pH 5.0 in acetate buffer with 0.02% (w/v) sodium azide added. Assays were run at 38° C. as a compromise between the higher activities of the cellulases at still higher temperatures and the temperature-tolerance of S. cerevisiae D5A, the organism used in companion simultaneous saccharification and fermentation (SSF) assays.
Pretreated corn stover (1% wt/wt sulfuric acid for 2-4 min at 190° C.) was prepared for use as a DSA substrate. Substrate loadings averaged 96.4 mg (dry wt.) biomass (standard deviation, n=5, of 0.8% or less) per DSA cell loading, for cellulose loadings of 55.5 mg glucose per assay. Substrate loadings thus amounted to 4.3% (w/v, solids) or 2.5% (w/v, cellulose). Because the effective saccharification of crystalline cellulose requires the synergistic action of both endoglucanases and exoglucanases, the activities of Cel7A and rCel7A proteins were assayed in combination with the catalytic domain of A. cellulolyticus endoglucanase I (EIcd), used here as a standard endoglucanase. In all DSA protocols, the particular Cel7A species being tested was loaded at 27.8 mg per g cellulose, and the endoglucanase (EIcd) was loaded at 1.11 mg per g cellulose, resulting in a 95:5 molar ratio of exoglucanase to endoglucanase. Quantitation of product sugars in effluent fractions was by HPLC using an Aminex HPX-87H column operated at 65° C. with 0.01 N H2SO4 (0.6 mL/min) as mobile phase, in an Agilent Model 1100 chromatograph.
Protein Stability Measurements
The overall protein stability of rCel7A from Example 4 was measured by differential scanning microcalorimetry using a Microcal model VP-DSC calorimeter (Microcal, Inc., Northampton, Mass.), with data analysis by means of Origin for DSC software (Microcal). Thermograms were collected for samples containing 50 μg/mL protein at pH 5.0 in 20 mM sodium acetate with 100 mM NaCl. Calorimeter scan rate was 60° C./h.
FIG. 5 shows comparison saccharification results as a plot of percent conversion of pretreated corn stover over time. Saccharification conditions included a pH of 5.0, a temperature of 38° C., and a standard enzyme loading of Cel7A at 27.8 mg/g cellulose plus EI endoglucanase at 1.13 mg/g cellulose. The comparison was between the specific performance of (A) native Cel7A from T. reesei, (B) native Cel7A from P. funiculosum, (C) rCel7A from P. funiculosum expressed in A. awamori, and (D) a one-half loading of rCel7A from P. funiculosum expressed in A. awamori. 
The results shown in FIG. 5 demonstrate advantages that are heretofore unknown in the art. Native T. reesei Cel7A is the current industry standard Cel7A enzyme used in cellulase formulations for biomass conversion. Surprisingly, an equivalent loading (27.8 mg protein/g cellulose) of purified Cel7A from P. funiculosum converted the pretreated corn stover substrate much more readily, with an approximate 69% conversion of cellulose being obtained at 72 hours, versus 52% conversion for the native T. reesei Cel7A. Furthermore, an equivalent loading of rCel7A from P. funiculosum expressed in A. awamori also yielded unexpectedly high performance, with 65% conversion of cellulose in 72 hours. This is important for process scale production of Cel7A, because heterologous expression of the P. funiculosum Cel7A from filamentous hosts is a viable large-scale production strategy. FIG. 5 also shows that at one-half loading (i.e., 13.9 mg protein/g cellulose) of the rCel7A P. funiculosum enzyme expressed in A. awamori, the performance on pretreated corn stover is still much greater than that of the native T. reesei Cel7A enzyme. These results show that the new P. funiculosum Cel7A+ A. cellulolyticus EIcd endoglucanase formulation deliver greater than 2-fold increase in specific performance relative to the T. reesei enzyme. Importantly, the native and recombinant Cel7A from P. funiculosum display this specific performance under conditions suitable for commercial SSF processes (pH 5 and 38° C.).
In conclusion, the data presented in FIG. 5 confirm that the cellulose digestion kinetics are such that native P. funiculosum Cel7A and P. funiculosum rCel7A expressed in A. awamori outperform native T. reesei Cel7A by a factor of at least two, as demonstrated by the superior performance of P. funiculosum rCel7A (13.9 mg protein/g cellulose) relative to T. reesei Cel7A (27.8 mg/g cellulose) at all points measured over the interval from 0 to 136 hours.
EXAMPLE 7
Characterization of P. funiculosum rCel7A
The purified rCel7A from Example 6 was tested against other Cel7A enzymes that were not expressed in A. awamori to determine the maximal thermal transition temperature. Test procedures included the use of differential scanning calorimetry (DSC) instrumentation.
FIG. 6 shows the comparative DSC results. The rCel7A from P. funiculosum expressed in A. awamori shows an optimal activation temperature of 66.8° C., as compared to 65.0° C. and 68.2° C., respectively, for native T. reesei Cel7A and rCel7A from T. reesei expressed in A. awamori. 
The foregoing discussion is intended to illustrate concepts by way of example with emphasis upon the preferred embodiments and instrumentalities. Accordingly, the disclosed embodiments and instrumentalities are not exhaustive of all options or mannerisms for practicing the disclosed principles hereof. The inventors hereby state their intention to rely upon the Doctrine of Equivalents in protecting the full scope and spirit of the invention.

## Claims

1. A method of producing a recombinant cellobiohydrolase 1 (rCel7A) enzyme, wherein the rCel7A enzyme has the amino acid sequence of SEQ ID NO: 8, comprising: obtaining a nucleic acid that encodes the amino acid sequence of SEQ ID NO 8 from Penicillium funiculosum, transforming a filamentous fungus with the nucleic acid; and culturing the filamentous fungus under conditions that produce the rCel7A enzyme in the filamentous fungus.

2. The method of claim 1, wherein the filamentous fungus used in the transforming and expressing steps comprises Aspergillus.

3. The method of claim 2, wherein the Aspergillus comprises Aspergillus awamori.

4. The method of claim 1, wherein the filamentous fungus used in the transforming and expressing steps comprises Trichoderma.

5. The method of claim 4, wherein the Trichoderma comprises Trichoderma reesei.

6. The method of claim 1, further comprising a step of isolating the rCel7A enzyme.

7. The method of claim 6, further comprising a step of mixing the rCel7A enzyme with an endoglucanase.

8. The method of claim 7, wherein the endoglucanase comprises Elcd endoglucanase.

9. The method of claim 8, wherein the step of mixing comprises mixing a 95:5 molar ratio of the rCel7A to Elcd endoglucanase.

10. A recombinant cellobiohydrolase 1 (rCel7A) enzyme having the amino acid sequence of SEQ ID NO: 8.

11. A recombinant cellobiohydrolase 1 (rCel7A) enzyme of claim 10, wherein the rCel7A enzyme is characterized by a peak activation temperature of 66.8° C.

12. A recombinant cellobiohydrolase 1 (rCel7A) enzyme from a filamentous fungus of the genus Penicillium, wherein the rCel7A enzyme has the amino acid sequence of SEQ ID NO: 8, wherein the rCel7A is produced by the method of claim 1.