Cloning and Expression of Full Length Trichoderma reesei Cellobiohydrolase I cDNAs in Escherichia coli

Robert A. Laymon; William S. Adney; Ali Mohagheghi; Michael E. Himmel; Steven R. Thomas

doi:10.1007/BF02941718

Cloning and Expression of Full Length Trichoderma reesei Cellobiohydrolase I cDNAs in Escherichia coli

Robert A. Laymon, William S. Adney, Ali Mohagheghi, Michael E. Himmel, Steven R. Thomas

BioEconomy and Sustainable Transportation

National Renewable Energy Laboratory

Research output: Contribution to journal › Article › peer-review

38 Scopus Citations

Abstract

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.

Original language	American English
Pages (from-to)	389-397
Number of pages	9
Journal	Applied Biochemistry and Biotechnology - Part A Enzyme Engineering and Biotechnology
Volume	57-58
DOIs	https://doi.org/10.1007/BF02941718 https://doi.org/10.1007/BF02941718
State	Published - 1996

NREL Publication Number

NREL/JA-422-7597

Keywords

Cellobiohydrolase I (CBHI)
Cellulases
Fusion proteins
T. reesei

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title = "Cloning and Expression of Full Length Trichoderma reesei Cellobiohydrolase I cDNAs in Escherichia coli",

abstract = "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.",

keywords = "Cellobiohydrolase I (CBHI), Cellulases, Fusion proteins, T. reesei",

author = "Laymon, {Robert A.} and Adney, {William S.} and Ali Mohagheghi and Himmel, {Michael E.} and Thomas, {Steven R.}",

year = "1996",

doi = "10.1007/BF02941718",

language = "American English",

volume = "57-58",

pages = "389--397",

journal = "Applied Biochemistry and Biotechnology - Part A Enzyme Engineering and Biotechnology",

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TY - JOUR

T1 - Cloning and Expression of Full Length Trichoderma reesei Cellobiohydrolase I cDNAs in Escherichia coli

AU - Laymon, Robert A.

AU - Adney, William S.

AU - Mohagheghi, Ali

AU - Himmel, Michael E.

AU - Thomas, Steven R.

PY - 1996

Y1 - 1996

N2 - 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.

AB - 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.

KW - Cellobiohydrolase I (CBHI)

KW - Cellulases

KW - Fusion proteins

KW - T. reesei

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U2 - 10.1007/BF02941718

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AN - SCOPUS:0030096896

SN - 0273-2289

VL - 57-58

SP - 389

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JO - Applied Biochemistry and Biotechnology - Part A Enzyme Engineering and Biotechnology

JF - Applied Biochemistry and Biotechnology - Part A Enzyme Engineering and Biotechnology

ER -

Cloning and Expression of Full Length Trichoderma reesei Cellobiohydrolase I cDNAs in Escherichia coli

Abstract

NREL Publication Number

Keywords

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