Chemistry and Chemical Biology ETDs

Publication Date

Spring 2023

Abstract

Humans have turned to natural products for medicinal therapeutics for millennia. In recent years, the ribosomally synthesized and post-translationally modified peptide (RiPP) class of natural products have garnered a lot of attention due to their ability for heterologous expression, relative ease of mutation, and vast biological activates. Microviridins are a family of RiPPs that contain a tricyclic cage-like structure through the formation of two lactone rings and one lactam ring catalyzed by two ATP-grasp ligase family enzymes. Microviridins are potent inhibitors of serine proteases and mutational studies of a small number of residues indicates the potential of peptide engineering protease inhibition. The screening of large libraries of microviridin mutants could allow for targeted protease inhibition.

Here we describe work toward the yeast surface display of microviridin B. It is demonstrated that Microviridin B retains its elastase inhibitory activity with an nM IC50 when the leader region of the precursor peptide is attached, opening the door to the surface display of the peptide for library screening.

An assay was developed screen microviridin B libraries to determine the specificity of APT grasp ligase MdnC. The proof of concept showed promising results, however there is indication of incomplete peptide modification by MdnC. An investigation into MdnC expression in yeast shows a 2µ origin and constitutive GPD promoter results in higher MdnC expression compared to a CEN origin and Gal1 promoter, even on galactose media.


Keywords

microviridin, natural products, RiPP, yeast surface display, protease, elastase

Document Type

Dissertation

Degree Name

Chemistry

Level of Degree

Doctoral

Department Name

Department of Chemistry and Chemical Biology

First Committee Member (Chair)

Mark C. Walker

Second Committee Member

Brian Gold

Third Committee Member

William Sherman Garver

Fourth Committee Member

Amy S. Gardiner

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