Expression of Talaromyces emersonii cellobiohydrolase Cel7A in Saccharomyces cerevisiae and rational mutagenesis to improve its thermostability and activity

Sanni Voutilainen, P. G. Murray, M. G. Tuohy, Anu Koivula (Corresponding Author)

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Abstract

We report here a successful expression of a single-module GH-7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (Te Cel7A) in Saccharomyces cerevisiae. The heterologous expression system allowed structure-guided protein engineering to improve the thermostability and activity of Te Cel7A. Altogether six different mutants aimed at introducing additional disulphide bridges to the catalytic module of Te Cel7A were designed. These included addition of five individual S-S bridges in or between the loops extending from the β-sandwich fold, and located either near the active site tunnel or forming the tunnel in Te Cel7A. A triple mutant containing the three best S-S mutations was also engineered. Three out of five single S-S mutants all had clearly improved thermostability which was also reflected as improved Avicel hydrolysis efficiency at 75°C. The best mutant was the triple mutant whose unfolding temperature was improved by 9°C leading to efficient microcrystalline cellulose hydrolysis at 80°C. All the additional S-S bonds contributed mainly to the thermostability of the Te Cel7A, but one of the mutants (N54C/P191C) also showed, somewhat surprisingly, improved activity even at room temperature.
Original languageEnglish
Pages (from-to)69-79
Number of pages11
JournalProtein Engineering, Design and Selection
Volume23
Issue number2
DOIs
Publication statusPublished - 2010
MoE publication typeA1 Journal article-refereed

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Talaromyces
Cellulose 1,4-beta-Cellobiosidase
Mutagenesis
Yeast
Saccharomyces cerevisiae
Hydrolysis
Tunnels
Protein Engineering
Temperature
Fungi
Cellulose
Disulfides
Catalytic Domain
Proteins
Mutation

Keywords

  • Cellulase
  • Disulphide bridge
  • Saccharomyces cerevisiae
  • Site-directed mutagenesis
  • Talaromyces emersonii

Cite this

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title = "Expression of Talaromyces emersonii cellobiohydrolase Cel7A in Saccharomyces cerevisiae and rational mutagenesis to improve its thermostability and activity",
abstract = "We report here a successful expression of a single-module GH-7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (Te Cel7A) in Saccharomyces cerevisiae. The heterologous expression system allowed structure-guided protein engineering to improve the thermostability and activity of Te Cel7A. Altogether six different mutants aimed at introducing additional disulphide bridges to the catalytic module of Te Cel7A were designed. These included addition of five individual S-S bridges in or between the loops extending from the β-sandwich fold, and located either near the active site tunnel or forming the tunnel in Te Cel7A. A triple mutant containing the three best S-S mutations was also engineered. Three out of five single S-S mutants all had clearly improved thermostability which was also reflected as improved Avicel hydrolysis efficiency at 75°C. The best mutant was the triple mutant whose unfolding temperature was improved by 9°C leading to efficient microcrystalline cellulose hydrolysis at 80°C. All the additional S-S bonds contributed mainly to the thermostability of the Te Cel7A, but one of the mutants (N54C/P191C) also showed, somewhat surprisingly, improved activity even at room temperature.",
keywords = "Cellulase, Disulphide bridge, Saccharomyces cerevisiae, Site-directed mutagenesis, Talaromyces emersonii",
author = "Sanni Voutilainen and Murray, {P. G.} and Tuohy, {M. G.} and Anu Koivula",
year = "2010",
doi = "10.1093/protein/gzp072",
language = "English",
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pages = "69--79",
journal = "Protein Engineering, Design and Selection",
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TY - JOUR

T1 - Expression of Talaromyces emersonii cellobiohydrolase Cel7A in Saccharomyces cerevisiae and rational mutagenesis to improve its thermostability and activity

AU - Voutilainen, Sanni

AU - Murray, P. G.

AU - Tuohy, M. G.

AU - Koivula, Anu

PY - 2010

Y1 - 2010

N2 - We report here a successful expression of a single-module GH-7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (Te Cel7A) in Saccharomyces cerevisiae. The heterologous expression system allowed structure-guided protein engineering to improve the thermostability and activity of Te Cel7A. Altogether six different mutants aimed at introducing additional disulphide bridges to the catalytic module of Te Cel7A were designed. These included addition of five individual S-S bridges in or between the loops extending from the β-sandwich fold, and located either near the active site tunnel or forming the tunnel in Te Cel7A. A triple mutant containing the three best S-S mutations was also engineered. Three out of five single S-S mutants all had clearly improved thermostability which was also reflected as improved Avicel hydrolysis efficiency at 75°C. The best mutant was the triple mutant whose unfolding temperature was improved by 9°C leading to efficient microcrystalline cellulose hydrolysis at 80°C. All the additional S-S bonds contributed mainly to the thermostability of the Te Cel7A, but one of the mutants (N54C/P191C) also showed, somewhat surprisingly, improved activity even at room temperature.

AB - We report here a successful expression of a single-module GH-7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (Te Cel7A) in Saccharomyces cerevisiae. The heterologous expression system allowed structure-guided protein engineering to improve the thermostability and activity of Te Cel7A. Altogether six different mutants aimed at introducing additional disulphide bridges to the catalytic module of Te Cel7A were designed. These included addition of five individual S-S bridges in or between the loops extending from the β-sandwich fold, and located either near the active site tunnel or forming the tunnel in Te Cel7A. A triple mutant containing the three best S-S mutations was also engineered. Three out of five single S-S mutants all had clearly improved thermostability which was also reflected as improved Avicel hydrolysis efficiency at 75°C. The best mutant was the triple mutant whose unfolding temperature was improved by 9°C leading to efficient microcrystalline cellulose hydrolysis at 80°C. All the additional S-S bonds contributed mainly to the thermostability of the Te Cel7A, but one of the mutants (N54C/P191C) also showed, somewhat surprisingly, improved activity even at room temperature.

KW - Cellulase

KW - Disulphide bridge

KW - Saccharomyces cerevisiae

KW - Site-directed mutagenesis

KW - Talaromyces emersonii

U2 - 10.1093/protein/gzp072

DO - 10.1093/protein/gzp072

M3 - Article

VL - 23

SP - 69

EP - 79

JO - Protein Engineering, Design and Selection

JF - Protein Engineering, Design and Selection

SN - 1741-0126

IS - 2

ER -