Cloning and expression of full-length Trichoderma reesei cellobiohydrolase I cDNAs in Escherichia coli

• Published in: Applied biochemistry and biotechnology 1996, Vol.57, p.389-398
• Main Authors: Laymon, R.A. (National Renewable Energy Laboratory, Golden, CO.), Adney, W.S, Mohagheghi, A, Himmel, M.E, Thomas, S.R
• Format: Article
• Language: English
• Subjects: Base Sequence; Biological and medical sciences; Biotechnology; Cellulase - biosynthesis; Cellulase - genetics; Cellulose 1,4-beta-Cellobiosidase; CLONACION; CLONAGE; Cloning, Molecular; DNA, Complementary - biosynthesis; ESCHERICHIA COLI; Fundamental and applied biological sciences. Psychology; Gene Expression; Gene Library; Genetic engineering; Genetic technics; Methods. Procedures. Technologies; Modification of gene expression level; Molecular Sequence Data; Recombinant Proteins - biosynthesis; RNA, Messenger - isolation & purification; Thioredoxins - biosynthesis; Thioredoxins - genetics; TRICHODERMA
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/BF02941718

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