Chemistry and Chemical Biology ETDs

Publication Date

Fall 11-15-2016

Abstract

Actinobacteria, especially those of genus Streptomyces, are a prominent source of bioactive natural products. The ability to site-specifically incorporate unnatural amino acids (UAAs) into natural product biosynthetic enzymes and ribosomally derived peptides in these organisms would constitute a valuable tool for drug discovery and development. The work described in this dissertation focuses on development and application of an expanded Streptomyces genetic code, including development of UAA incorporation systems based on amber suppression and sense codon reassignment, structural diversification of the model thiopeptide natural product thiostrepton using UAAs, and mapping protein-protein interactions in type II polyketide biosynthetic enzymes using photocrosslinking UAAs.

First, we developed an amber suppression-based system of site-specific incorporation of p-iodo-L-phenylalanine (pIPhe) and p-azido-L-phenylalanine (pAzPhe) into superfolder GFP (sfGFP) in the model natural product producer Streptomyces venezuelae ATCC 15439.

Next, the rare leucine codon TTA was reassigned to encode pIPhe and p-benzoyl-L-phenylalanine (pBpa) in S. coelicolor J1681 (ΔbldA), in which the unique tRNALeuUAA (bldA) that recognizes the TTA codon was deleted. In the S. venezuelae ΔbldA strain, we achieved 20-fold higher yields of UAA containing protein using the TTA reassignment system compared to the amber suppression-based system; and were able to incorporate up to 10 scattered or 5 tandem UAAs in a single protein using TTA reassignment.

Finally, we have carried out preliminary work on two applications. In the first, we constructed and tested functionality of a system designed to incorporate pAzPhe into the actinorhodin ketosynthase β (KSβ) in S. coelicolor J1681 to interrogate protein-protein interactions in actinorhodin biosynthesis. In the second, we have begun developing a system for incorporation of UAAs into thiostrepton in the native producer Streptomyces laurentii ATCC 31255. Preliminary results confirm the functionality of amber suppression system in S. laurentii; and demonstrated development of a TipA-based fluorescent biosensor for detecting thiopeptide antibiotics in S. venezuelae. Work on these two applications has laid the foundation for development of tools to structurally diversify the ribosomally synthesized peptides and to address questions related to natural product biosynthesis and mechanism of action that are relevant to drug discovery and development.

Project Sponsors

UNM and NM-INBRE

Language

English

Keywords

unnatural amino acids, Streptomyces, p-azido-L-phenylalanine, p-iodo-L-phenylalanine, natural products

Document Type

Dissertation

Degree Name

Chemistry

Level of Degree

Doctoral

Department Name

Department of Chemistry and Chemical Biology

First Committee Member (Chair)

Charles E. Melançon III

Second Committee Member

Debra Dunaway-Mariano

Third Committee Member

Fu-sen Liang

Fourth Committee Member

Jeremy S. Edwards

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