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Noncanonical Amino Acid Synthesis by Evolved Tryptophan Synthases


Almhjell, Patrick James (2023) Noncanonical Amino Acid Synthesis by Evolved Tryptophan Synthases. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/rcvq-dg51.


The β-subunit of tryptophan synthase (TrpB) is responsible for the final step of ʟ-tryptophan biosynthesis in all of known biology. Recognized for this important role and its powerful chemistry, TrpB has more recently been used for the in vitro synthesis of tryptophan analogs and other noncanonical amino acids (ncAAs). This thesis describes some of these efforts as well as the application of TrpB for developing new methods in directed enzyme evolution. Chapter I first establishes important topical background. It begins with a general account of directed enzyme evolution by exploring the emergence of new catalytic functions in natural and laboratory settings, and how this information and chemical intuition can be used to create enzymes for desired reactions. This is followed by a description of the state of the field of ncAA synthesis, with a special focus on biocatalytic approaches using engineered enzymes. Chapters II and III examine targeted engineering campaigns to create enzymes that can efficiently synthesize valuable blue-fluorescent ncAAs such as 4-cyanotryptophan (Chapter II) and β-(1-azulenyl)-ʟ-alanine (AzAla, Chapter III) from accessible starting materials. In Chapter IV, the native function of TrpB for ʟ-tryptophan biosynthesis is used as a selection pressure to develop an in vivo continuous evolution system. Despite a selection pressure for only ʟ-tryptophan synthesis the orthologous TrpB variants generated by this system have varying promiscuous activities for ʟ-tryptophan analogs, paralleling the sequence-function diversity of natural enzyme homologs. Chapter V describes evSeq, an inexpensive and simple method for sequencing all protein variants generated during an engineering campaign, demonstrated by collecting ~800 TrpB sequence-function data points. Finally, in Chapter VI, directed evolution and chemical intuition are used to convert TrpB from a tryptophan synthase to a novel tyrosine synthase (TyrS). This enzyme can irreversibly and regioselectively alkylate simple phenol analogs to synthesize valuable tyrosine analogs, including the blue-fluorescent ncAA β-(1-naphthol-4-yl)-ʟ-alanine (NaphAla) and 3-methyl-ʟ-tyrosine at gram scales. Because TyrS synthesizes a primary metabolite, this transformation represents a noncanonical method for the biosynthesis of ʟ-tyrosine. This is the first example of a feasible new route for de novo aromatic amino acid biosynthesis, which occurs through a universally conserved set of chemistry across all of life. In total, the work described here expands the fields of chemical synthesis and synthetic biology by presenting new enzymes—and methods for producing these enzymes—that are capable of synthesizing important amino acids in vitro and in vivo.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:noncanonical amino acids; biocatalysis; directed evolution; enzyme engineering; tryptophan synthase; tyrosine; C–C bond formation;
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Arnold, Frances Hamilton
Thesis Committee:
  • Wang, Kaihang (chair)
  • Fu, Gregory C.
  • Mayo, Stephen L.
  • Arnold, Frances Hamilton
Defense Date:11 October 2022
Funding AgencyGrant Number
National Institute of General Medical Sciences (NIGMS)R01GM125887
Dr. Nagendranath Reddy Graduate FellowshipUNSPECIFIED
Record Number:CaltechTHESIS:10172022-224813021
Persistent URL:
Related URLs:
URLURL TypeDescription adapted for Chapter I adapted for Chapter II adapted for Chapter III adapted for Chapter IV adapted for Chapter V
Almhjell, Patrick James0000-0003-0977-841X
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:15042
Deposited By: Patrick Almhjell
Deposited On:25 Oct 2022 21:33
Last Modified:08 Nov 2023 00:11

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