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Engineering Enzyme Systems by Recombination


Trudeau, Devin Lee (2014) Engineering Enzyme Systems by Recombination. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/F8T8-3S80.


Homologous recombination is a source of diversity in both natural and directed evolution. Standing genetic variation that has passed the test of natural selection is combined in new ways, generating functional and sometimes unexpected changes. In this work we evaluate the utility of homologous recombination as a protein engineering tool, both in comparison with and combined with other protein engineering techniques, and apply it to an industrially important enzyme: Hypocrea jecorina Cel5a.

Chapter 1 reviews work over the last five years on protein engineering by recombination. Chapter 2 describes the recombination of Hypocrea jecorina Cel5a endoglucanase with homologous enzymes in order to improve its activity at high temperatures. A chimeric Cel5a that is 10.1 °C more stable than wild-type and hydrolyzes 25% more cellulose at elevated temperatures is reported. Chapter 3 describes an investigation into the synergy of thermostable cellulases that have been engineered by recombination and other methods. An engineered endoglucanase and two engineered cellobiohydrolases synergistically hydrolyzed cellulose at high temperatures, releasing over 200% more reducing sugars over 60 h at their optimal mixture relative to the best mixture of wild-type enzymes. These results provide a framework for engineering cellulolytic enzyme mixtures for the industrial conditions of high temperatures and long incubation times.

In addition to this work on recombination, we explored three other problems in protein engineering. Chapter 4 describes an investigation into replacing enzymes with complex cofactors with simple cofactors, using an E. coli enolase as a model system. Chapter 5 describes engineering broad-spectrum aldehyde resistance in Saccharomyces cerevisiae by evolving an alcohol dehydrogenase simultaneously for activity and promiscuity. Chapter 6 describes an attempt to engineer gene-targeted hypermutagenesis into E. coli to facilitate continuous in vivo selection systems.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:recombination; cellulase; biofuels; protein engineering; directed evolution; alcohol dehydrogenase; dehydratase; somatic hypermutagenesis; thermostability
Degree Grantor:California Institute of Technology
Division:Biology and Biological Engineering
Major Option:Bioengineering
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Arnold, Frances Hamilton
Thesis Committee:
  • Arnold, Frances Hamilton (chair)
  • Tirrell, David A.
  • Mayo, Stephen L.
  • Miller, Thomas F.
  • Shapiro, Mikhail G.
Defense Date:11 December 2013
Non-Caltech Author Email:devintrudeau (AT)
Funding AgencyGrant Number
Army Research Office--Institute for Collaborative BiotechnologiesW911NF-09-D-0001
National Science and Engineering Research Council PGSD3-404332-2011
Record Number:CaltechTHESIS:01202014-144143175
Persistent URL:
Related URLs:
URLURL TypeDescription adapted for ch. 1
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:8055
Deposited By: Devin Trudeau
Deposited On:24 Mar 2014 17:01
Last Modified:08 Nov 2023 00:11

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