CRADA Report

NREL/TP-7A1-48426 

August 2010

Clone Bacterial Thermal Stable 

Enzymes in T. Reesei

Cooperative Research and Development 

Final Report

CRADA Number: CRD-01-00105

NREL Technical Contact: Michael E. Himmel
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Cooperative Research and Development Final Report

In accordance with Requirements set forth in Article XI.A(3) of the CRADA document, this document is 

the final CRADA report, including a list of Subject Inventions, to be forwarded to the Office of Science

and Technical Information as part of the commitment to the public to demonstrate results of federally 

funded research.

CRADA number: CRD-01-00105

CRADA Title: Clone Bacterial Thermal Stable Enzymes in T. Reesei

Parties to the Agreement: Genencor Division, Danisco US, Inc.

Joint Work Statement Funding Table showing DOE commitment:

Estimated Costs NREL 

Shared Resources

Task 1

Loaded Labor $ 10,000.00

Other Costs $ 1,000.00

Task 2

Loaded Labor $ 170,000.00

Other Costs $ 19,000.00

Task 3

Loaded Labor $ 00.00

Other Costs $ 00.00

Task 4

 Loaded Labor

 Other Costs

 $ 90,000.00

 $ 10,000.00

TOTALS $ 300,000.00
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Abstract of CRADA work:

Cellulose, an unbranched β-1,4 linked homopolymer of glucose, is the most abundant renewable 

fuel resource on Earth, accounting for about half of the organic material in the biosphere, and is

the major polysaccharide found in plant biomass. The hydrolysis of cellulose, aided by 

endocellulase, exocellulase, and β-glucosidase catalysis, produces glucose, an easily fermentable 

monosaccharide. Intense research is aimed at the conversion of cellulose to ethanol because, as a 

source of renewable fuel, the process has great economic potential and is environmentally 

friendly. Cellulose is insoluble and crystalline, hence it is largely resistant to microbial attack. In 

many biomass utilization schemes the raw material is first treated with dilute acid at moderate 

temperatures to remove lignin and to speed up cellulose hydrolysis. The pretreated biomass can 

then be subjected to carefully chosen mixtures of endo- and exoglucanases for maximum cost 

effectiveness. 

Highly thermostable cellulase enzymes are known to be secreted by the cellulolytic thermophile 

Acidothermus cellulolyticus gen. nov., sp. nov., a bacterium originally isolated from decaying wood in 

an acidic, thermal pool at Yellowstone National Park (Mohagheghi et al. 1986. Int. J. System. 

Bacteriol. 36:435-443). The cellulase complex produced by this organism is known to contain several 

different cellulase enzymes with maximal activities at temperatures of 75o

C to 83oC. In addition to 

demonstrating the useful property of cellobiose (end product) inhibition resistance, the cellulases 

from A. cellulolyticus are active over a broad pH range centered about pH 5, the pH at which yeasts 

are capable of fermenting cellobiose and glucose to ethanol. A high molecular weight cellulase 

isolated from growth broths of A. cellulolyticus was found by SDS-PAGE of approximately 156,600 to 

203,400 daltons was described by U.S. patent #5,110,735. 

A novel cellulase enzyme, known as the E1 endoglucanase and also secreted by A. cellulolyticus into 

the growth medium, is described in detail in the U.S. Patent #5,275,944. The A. cellulolyticus E1 

endoglucanase is one of the most active cellulases known. In combination with the exocellulase 

CBH I from Trichoderma reesei, E1 gives the highest saccharification and degree of synergism of all 

cellulases tested at NREL. This useful endoglucanase demonstrates a temperature optimum of 83o

C 

and a specific activity of 40 μmole glucose release from carboxymethylcellulose/min/mg protein. E1 

endoglucanase was further identified as having an isoelectric pH of 6.7 and a molecular weight of 

81,000 daltons by SDS polyacrylamide gel electrophoresis. The gene coding EI catalytic and cellulose 

binding domains and linker peptide were protected by US Patent # 5,536,655. 

The NREL has achieved expression of active E1 catalytic domain in E. coli using a variety of 

expression vectors and this host is currently used for routine protein engineering work. Full length 

E1 is best expressed from Streptomyces lividans TK24, presumably because fewer serine proteases 

are produced by this most. NREL has tested a strain of Pichia pastoris designed for expression and 

secretion of the A. cellulolyticus E1 endoglucanase. In this construction, the mature E1 coding 

sequence was joined in the same translational reading frame to the yeast alpha factor signal 

sequence present in pPIC9. Transformants have not yet been analyzed for gene copy number, but 

unoptimized fermentations have already yielded 1.5 g/L of E1. This is by far the highest 

expression level for any strain yet developed at NREL for any gene. E1 has been expressed in an 

active and thermal stable enzyme from a variety of plants, including Arabidopsis, tobacco, cotton, 

barley, and duckweed. 
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NREL has recently identified and characterized a series of related and important glycosyl hydrolases 

produced by A. cellulolyticus. We expect that these high GC coding sequences will be similarly well 

expressed in a wide range of commercial hosts, but this is yet unproven. We also expect that all 

enzymes secreted from A. cellulolyticus will be thermal tolerant and synergistic with E1 and each 

other, as well as the known T. reesei cellulases. 

Summary of Research Results: 

The focus of this CRADA was on improving the efficacy of GCI’s primary host for commercial cellulase 

production, Trichoderma reesei. We employed a diversity of technologies in modern protein 

biotechnology to clone and express selected genes initially from the thermophilic bacterium, A. 

cellulolyticus, in experimental and commercial strains of T. reesei. As the work progressed, either 

partner identified other promising genes from the same or other bacterial sources that warranted 

expression research and these were considereded for inclusion in the project. 

Subject Inventions listing: None.

Report Date: 4/30/10 Responsible Technical Contact at Alliance/NREL: Himmel, Michael E.

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