Improving the thermal stability of cellobiohydrolase Cel7A from Hypocrea jecorina by directed evolution


Abstract

Secreted mixtures of Hypocrea jecorina cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. H. jecorina Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion. Here, we enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and a Cel7A variant (FCA398) was obtained, which exhibited a 10.4 °C increase in Tm and a 44-fold greater half-life compared with the wild-type enzyme. This Cel7A variant contains 18 mutated sites and is active under application conditions up to at least 75 °C. The X-ray crystal structure of the catalytic domain was determined at 2.1 Å resolution and showed that the effects of the mutations are local and do not introduce major backbone conformational changes. Molecular dynamics simulations revealed that the catalytic domain of wild-type Cel7A and the FCA398 variant exhibit similar behavior at 300 K, whereas at elevated temperature (475 and 525 K), the FCA398 variant fluctuates less and maintains more native contacts over time. Combining the structural and dynamic investigations, rationales were developed for the stabilizing effect at many of the mutated sites.

Submission Details

ID: UQuoyfyA

Submitter: Shu-Ching Ou

Submission Date: Oct. 11, 2018, 10:32 a.m.

Version: 1

Publication Details
Goedegebuur F;Dankmeyer L;Gualfetti P;Karkehabadi S;Hansson H;Jana S;Huynh V;Kelemen BR;Kruithof P;Larenas EA;Teunissen PJM;Ståhlberg J;Payne CM;Mitchinson C;Sandgren M,J Biol Chem (2017) Improving the thermal stability of cellobiohydrolase Cel7A from <i>Hypocrea jecorina</i> by directed evolution. PMID:28860192
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2CBH 1990-01-15 DETERMINATION OF THE THREE-DIMENSIONAL STRUCTURE OF THE C-TERMINAL DOMAIN OF CELLOBIOHYDROLASE I FROM TRICHODERMA REESEI. A STUDY USING NUCLEAR MAGNETIC RESONANCE AND HYBRID DISTANCE GEOMETRY-DYNAMICAL SIMULATED ANNEALING
1AZJ 1998-04-29 THREE-DIMENSIONAL STRUCTURES OF THREE ENGINEERED CELLULOSE-BINDING DOMAINS OF CELLOBIOHYDROLASE I FROM TRICHODERMA REESEI, NMR, 18 STRUCTURES
5X36 2017-05-31 Solution structure of the Family 1 carbohydrate-binding module with mannosylated Ser3
1AZK 1998-04-29 THREE-DIMENSIONAL STRUCTURES OF THREE ENGINEERED CELLULOSE-BINDING DOMAINS OF CELLOBIOHYDROLASE I FROM TRICHODERMA REESEI, NMR, 19 STRUCTURES
5X38 2017-05-31 Solution structure of the Family 1 carbohydrate-binding module with glucosylated Ser3
5X35 2017-05-31 Solution structure of the Family 1 carbohydrate-binding module with mannosylated Thr1
5X39 2017-05-31 Solution structure of the Family 1 carbohydrate-binding module Q2A mutant with mannosylated Ser3
1CBH 1990-01-15 DETERMINATION OF THE THREE-DIMENSIONAL STRUCTURE OF THE C-TERMINAL DOMAIN OF CELLOBIOHYDROLASE I FROM TRICHODERMA REESEI. A STUDY USING NUCLEAR MAGNETIC RESONANCE AND HYBRID DISTANCE GEOMETRY-DYNAMICAL SIMULATED ANNEALING
5X3C 2017-05-31 Solution structure of the Family 1 carbohydrate-binding module Y5A mutant with mannosylated Ser3
1AZ6 1998-04-08 THREE-DIMENSIONAL STRUCTURES OF THREE ENGINEERED CELLULOSE-BINDING DOMAINS OF CELLOBIOHYDROLASE I FROM TRICHODERMA REESEI, NMR, 23 STRUCTURES
5X37 2017-05-31 Solution structure of the Family 1 carbohydrate-binding module with mannosylated Ser14
5X34 2017-05-31 Solution structure of the Family 1 carbohydrate-binding module, unglycosylated form
2MWK 2015-09-02 Family 1 Carbohydrate-Binding Module from Trichoderma reesei Cel7A with O-mannose residues at Thr1, Ser3, and Ser14
1AZH 1998-04-08 THREE-DIMENSIONAL STRUCTURES OF THREE ENGINEERED CELLULOSE-BINDING DOMAINS OF CELLOBIOHYDROLASE I FROM TRICHODERMA REESEI, NMR, 14 STRUCTURES
2MWJ 2015-09-02 Solution structure of Family 1 Carbohydrate-Binding Module from Trichoderma reesei Cel7A with O-mannose residues at Thr1 and Ser3
2V3I 2008-07-01 1.05 Hypocrea jecorina Cel7A in complex with (R)-dihydroxy-phenanthrenolol
4UWT 2015-08-26 1.2 Hypocrea jecorina Cel7A E212Q mutant in complex with p-nitrophenyl cellobioside
4C4D 2014-01-08 1.32 Covalent glycosyl-enzyme intermediate of Hypocrea jecorina Cel7a E217Q mutant trapped using DNP-2-deoxy-2-fluoro-cellotrioside
4D5I 2015-03-25 1.42 Hypocrea jecorina cellobiohydrolase Cel7A E212Q soaked with xylotriose.
4C4C 2014-01-08 1.45 Michaelis complex of Hypocrea jecorina CEL7A E217Q mutant with cellononaose spanning the active site
4D5J 2015-03-25 1.5 Hypocrea jecorina cellobiohydrolase Cel7A E217Q soaked with xylotriose.
4P1H 2015-03-04 1.5 Crystal structure of wild type Hypocrea jecorina Cel7a in a monoclinic crystal form
4D5O 2015-03-25 1.52 Hypocrea jecorina cellobiohydrolase Cel7A E212Q soaked with xylopentaose.
1Q2B 2003-11-25 1.6 CELLOBIOHYDROLASE CEL7A WITH DISULPHIDE BRIDGE ADDED ACROSS EXO-LOOP BY MUTATIONS D241C AND D249C
2V3R 2008-07-01 1.6 Hypocrea jecorina Cel7A in complex with (S)-dihydroxy-phenanthrenolol
1EGN 2001-05-16 1.6 CELLOBIOHYDROLASE CEL7A (E223S, A224H, L225V, T226A, D262G) MUTANT
4D5V 2015-03-25 1.62 Hypocrea jecorina cellobiohydrolase Cel7A E217Q soaked with xylotetraose.
4D5Q 2015-03-25 1.68 Hypocrea jecorina Cel7A (wild type) soaked with xylopentaose.
4V0Z 2015-09-30 1.7 o-nitrophenyl Cellobioside as an Active Site Probe for Family 7 Cellobiohydrolases
6CEL 1997-12-24 1.7 CBH1 (E212Q) CELLOPENTAOSE COMPLEX
1Q2E 2003-11-25 1.75 CELLOBIOHYDROLASE CEL7A WITH LOOP DELETION 245-252 AND BOUND NON-HYDROLYSABLE CELLOTETRAOSE
6GRN 2018-10-10 1.79 CELLOBIOHYDROLASE I (CEL7A) FROM Trichoderma reesei with S-dihydroxypropranolol in the active site
1CEL 1994-11-01 1.8 THE THREE-DIMENSIONAL CRYSTAL STRUCTURE OF THE CATALYTIC CORE OF CELLOBIOHYDROLASE I FROM TRICHODERMA REESEI
4D5P 2015-03-25 1.89 Hypocrea jecorina cellobiohydrolase Cel7A E217Q soaked with xylopentaose.
7CEL 1997-12-24 1.9 CBH1 (E217Q) IN COMPLEX WITH CELLOHEXAOSE AND CELLOBIOSE
5CEL 1997-12-24 1.9 CBH1 (E212Q) CELLOTETRAOSE COMPLEX
1DY4 2000-12-18 1.9 CBH1 IN COMPLEX WITH S-PROPRANOLOL
3CEL 1997-03-12 2.0 ACTIVE-SITE MUTANT E212Q DETERMINED AT PH 6.0 WITH CELLOBIOSE BOUND IN THE ACTIVE SITE
2CEL 1997-03-12 2.0 ACTIVE-SITE MUTANT E212Q DETERMINED AT PH 6.0 WITH NO LIGAND BOUND IN THE ACTIVE SITE
5OA5 2017-09-06 2.1 CELLOBIOHYDROLASE I (CEL7A) FROM HYPOCREA JECORINA WITH IMPROVED THERMAL STABILITY
4CEL 1997-03-12 2.2 ACTIVE-SITE MUTANT D214N DETERMINED AT PH 6.0 WITH NO LIGAND BOUND IN THE ACTIVE SITE
4P1J 2015-03-25 2.62 Crystal structure of wild type Hypocrea jecorina Cel7a in a hexagonal crystal form

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.1 Exoglucanase 1 P19355 GUX1_HYPRU
97.7 Exoglucanase 1 A0A024RXP8 GUX1_HYPJR
100.0 Exoglucanase 1 P62695 GUX1_TRIKO
100.0 Exoglucanase 1 P62694 GUX1_HYPJE