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Directed Evolution of Terpene Synthases for Non-Natural Substrates


Kalbarczyk, Karolina Zbigniew (2013) Directed Evolution of Terpene Synthases for Non-Natural Substrates. Senior thesis (Major), California Institute of Technology. doi:10.7907/N4T5-FP16.


Terpenes represent about half of known natural products, with terpene synthases catalyzing reactions to increase the complexity of substrates and generate cyclizations of the linear diphosphate substrates, therefore forming rings and stereocenters. With their diverse functionality, terpene synthases may be highly evolvable, with the ability to accept a wide range of non-natural compounds and with high product selectivity. Our hypothesis is that directed evolution of terpene synthases can be used to increase selectivity of the synthase on a specific substrate. In the first part of the work presented herein, three natural terpene synthases, Cop2, BcBOT2, and SSCG_02150, were tested for activity against the natural substrate and a non-natural substrate, called Surrogate 1, and the relative activities on both the natural and non-natural substrates were compared. In the second part of this work, a terpene synthase variant of BcBOT2 that has been evolved for thermostability, was used for directed evolution for increased activity and selectivity on the non-natural substrate referred to as Surrogate 2. Mutations for this evolution were introduced using random mutagenesis, with error prone polymerase chain reactions, and using site-specific saturation mutagenesis, in which an NNK library is designed with a specific active site amino acid targeted for mutation. The mutant enzymes were then screened and selected for enhancement of the desired functionality. Two neutral mutants, 19B7 W367F and 19B7 W118Q, were found to maintain activity on Surrogate 2, as measured by the screen.

Item Type:Thesis (Senior thesis (Major))
Subject Keywords:terpene; synthase; synthases; terpenoid; terpenoids; directed; evolution; non-natural substrates; terpene synthase; terpene synthases; karolina kalbarczyk;
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemical Engineering
Awards:Library Friends Senior Thesis Prize Winner, 2013.
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Arnold, Frances Hamilton (advisor)
  • Lauchli, Ryan (co-advisor)
Group:Senior Undergraduate Thesis Prize
Thesis Committee:
  • Arnold, Frances Hamilton (chair)
Defense Date:April 2013
Funding AgencyGrant Number
California Institute of TechnologyUNSPECIFIED
Summer Undergraduate Research FellowshipUNSPECIFIED
Record Number:CaltechTHESIS:05302013-204117395
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7779
Deposited By: Karolina Kalbarczyk
Deposited On:31 May 2013 18:58
Last Modified:08 Nov 2023 00:11

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