Cloning and expression of full-length Trichoderma reesei cellobiohydrolase I cDNAs in Escherichia coli.
The process of converting lignocellulosic biomass to ethanol via fermentation depends on developing economic sources of cellulases. Trichoderma reesei cellobiohydrolase (CBH) I is a key enzyme in the fungal cellulase system; however, specific process application requirements make modification of the enzyme by site-directed mutagenesis (SDM) an attractive goal. To undertake SDM investigations, an efficient, cellulase-free host is required. To test the potential of Escherichia coli as a host, T. reesei CBH I cDNA was expressed in E. coli strain GI 724 as a C-terminal fusion to thermostable thioredoxin protein. Full-length expression of CBH I was subsequently verified by molecular weight, Western blot analysis, and activity on soluble substrates.
Laymon
R A
RA
Applied Biological Sciences Branch, Alternative Fuels Division, National Renewable Energy Laboratory, Golden, CO 80401, USA.
Adney
W S
WS
Mohagheghi
A
A
Himmel
M E
ME
Thomas
S R
SR
D001483:Base Sequence; D002480:Cellulase / Q000096:biosynthesis / Q000235:genetics*; D043366:Cellulose 1,4-beta-Cellobiosidase; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis*; D004926:Escherichia coli; D015870:Gene Expression; D015723:Gene Library; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis; D013879:Thioredoxins / Q000096:biosynthesis / Q000235:genetics*; D014242:Trichoderma
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013879:Thioredoxins; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase