Investigation of the Tailoring Steps in Pradimicin Biosynthesis

Author

Kandy L. Napan, Utah State UniversityFollow

Date of Award:

5-2016

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biological Engineering

Committee Chair(s)

Jixun Zhan (Committee Chair), Jon Y. Takemoto (Committee Co-Chair)

Committee

Jixun Zhan

Committee

Jon Y. Takemoto

Committee

Cheng-Wei Tom Chang

Committee

Michelle Grilley

Committee

Charles Miller

Abstract

This research focused on the investigation of the late steps in the biosynthetic pathway of the novel antifungal and antiviral pradimicins A-C. Pradimicins were first isolated from the soil bacterium Actinomadura hibisca. These bioactive molecules are assembled by a type II polyketide biosynthetic pathway. Although the biosynthetic gene cluster of pradimicin has been identified, the functions of the biosynthetic genes and how they work collaboratively to form the final structures of pradimicins remain unknown. This research aims to functionally characterize the enzymes involved in the late steps of the biosynthetic route.

The early biosynthetic steps of pradimicins have been previously investigated, and the enzymes required to form the core pentangular structure have been identified. This dissertation research characterized eight biosynthetic enzymes that further tailor the core structure. These enzymes include PdmJ, PdmW, PdmN, PdmT, PdmO, PdmS, PdmQ and PdmF. Main research approaches included gene disruption, heterologous expression, combinatorial biosynthesis, and in vitro biochemical studies. PdmJ and PdmW were characterized as cytochrome P450 hydroxylases that catalyze the incorporation of two hydroxyl groups at C-5 and C-6, respectively. These enzymes worked synergistically during pradimicin biosynthesis. PdmN is an amino acid ligase with broad substrate specificity. PdmS and PdmQ were identified as the dedicated O-glycosyltransferases that are responsible for the introduction of the first and second sugar moieties. Three methyltransferases involved in pradimicin biosynthesis were also identified. PdmO is a Nmethyltransferase responsible for the methylation of the amino sugar moiety. PdmF was identified as the C-11 O-methyltransferase, while PdmT was confirmed to be an Omethyltransferase that methylates the 7-OH.

Engineered biosynthesis has shown its promise in creating chemical diversity for drug discovery. This research has identified eight critical biosynthetic enzymes in the biosynthesis of pradimicins, a group of promising antiviral and antifungal compounds. The results from this work provide an important knowledge and technical basis for further engineering of this pathway to generate new pradimicin analogues for bioactivity screening.

Checksum

6ed3bc0a70b0ad1eda11738de4d58f0a

Recommended Citation

Napan, Kandy L., "Investigation of the Tailoring Steps in Pradimicin Biosynthesis" (2016). All Graduate Theses and Dissertations, Spring 1920 to Summer 2023. 4730.
https://digitalcommons.usu.edu/etd/4730