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Title: EXPRESSION AND CHARACTERIZATION OF ACIDOTHERMUS CELLULOLYTICUS E1 ENDOGLUCANASE IN TRANSGENIC DUCKWEED LEMNA MINOR 8627

Author
item SUN, YE - NC STATE UNIV
item CHENG, JAY - NC STATE UNIV
item HIMMEL, MICHAEL - NAT RENEW ENERGY LAB
item Skory, Christopher - Chris
item ADNEY, WILLIAM - NAT RENEW ENERGY LAB
item THOMAS, STEVEN - NAT RENEW ENERGY LAB
item Tisserat, Brent
item NISHIMURA, YUFUKO - NC STATE UNIV
item YAMAMOTO, YURI - NC STATE UNIV

Submitted to: Bioresource Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/28/2006
Publication Date: 12/15/2006
Citation: Sun, Y., Cheng, J.J., Himmel, M.E., Skory, C.D., Adney, W.S., Thomas, S.R., Tisserat, B., Nishimura, Y., Yamamoto, Y.T. 2006. Expression and characterization of Acidothermus cellulolyticus E1 endoglucanase in transgenic duckweed Lemna minor 8627. Bioresource Technology. Available: http://www.sciencedirect.com, doi:10.1016/j.biortech.2006.09.055

Interpretive Summary: Agricultural biomass is composed primarily of sugars linked together to form the polymer more commonly known as cellulose. This abundant plant product offers great potential as a renewable resource for the production of numerous commodity chemicals. Converting the cellulose back into simple sugars using enzymes provides a feedstock that can be used for microbial fermentations that produce high value products such as fuel ethanol. However, producing these enzymes in large quantities is typically cost prohibitive to the process. Therefore, we have developed novel methods to express one of these enzymes in a fast growing aquatic plant called Lemna. This technology will allow the Lemna to be grown at the conversion facilities in contained basins with minimal capital expenditure. We anticipate that continued improvements with this system could lead to significant reduction in production costs for numerous enzymes. This work initially will benefit primarily industries requiring enzymes, corn processors, and fuel ethanol manufacturers.

Technical Abstract: Endoglucanase E1 from Acidothermus cellulolyticus was expressed cytosolically under control of the cauliflower mosaic virus 35S promoter in transgenic duckweed, Lemna minor 8627. The selected transgenic duckweed line Cel25IX-15 generated using Agrobacterium-mediated transformation exhibited no phenotypic or growth abnormality. Immunodetection of whole plant extracts revealed that enzyme co-migrated with purified catalytic domain fraction obtained by papain treatment of the secreted native E1 protein. Enzyme produced by isolate Cel25IX-15 was biologically active and the expression level was 0.24% of total soluble protein. The endoglucanase activity with carboxymethylcellulose was 0.24 umol glucose g**-1 fresh duckweed min**-1 at 65°C. The optimal temperature and pH for E1 enzyme activity were about 80°C and pH 5, respectively. Among three extraction buffers tested, HEPES (N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) buffer (pH 8) extracted the most E1 enzyme and total soluble proteins from the transgenic duckweed. Heat treatment at 65°C during the crude extract preparation effectively removed most of other soluble proteins and slightly increased the extracted CMC-degrading activity. Citrate buffer (pH 4.8) in conjunction with the heat treatment enriched relative amounts of E1 enzyme in the extract. This study demonstrates that duckweed may offer new options for the expression of cellulolytic enzymes in transgenic plants.