Date of Award:

12-2017

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biological Engineering

Committee Chair(s)

Jixun Zhan

Committee

Jixun Zhan

Committee

David W. Britt

Committee

Dong Chen

Committee

Jon Takemoto

Committee

Elizabeth Vargis

Abstract

This research is focused on investigation and engineering of natural product biosynthetic pathways for efficient production of pharmaceutically important molecules or generation of new bioactive molecules for drug development.

Natural products are an important source of therapeutics, such as chromomycin (anti-cancer), emodin (anti-inflammatory and anti-tumor) and sprolaxine (anti-Helicobacter pylori). Metabolic engineering of natural product biosynthetic pathways shows its promise for creating and producing valuable compounds with chemical diversity for drug discovery. One goal of this research is to create highly efficient strains to biosynthesize valuable natural products. The engineered Streptomyces roseiscleroticus strain constructed in this work showed higher titers of chromomycins than previously reported, which was achieved by characterizing and engineering the chromomycin biosynthetic gene cluster. I activated the polyketide biosynthetic pathway by engineering two regulatory genes, and optimized the culture conditions to increase the titer of chromomycins. The production of emodin nowadays mostly relies on conventional plant cultivation and organic solvent extraction, which is time-consuming and cost-ineffective. This work built a biosynthetic platform using industrial strains Saccharomyces cerevisiae and Pichia pastoris with eight genes from fungi and yeast, which affords a more efficient biosynthetic process of emodin.

On the other hand, we used Escherichia coli as a platform for heterologous expression of PKSs and engineering of particular biosynthetic pathways to generate chemical diversity in natural products. The type III polyketide synthase (PKS) involved in the biosynthesis of spirolaxine was identified in this research, which is important for complete elucidation of the biosynthetic pathway of this anti-Helicobacter pylori natural product. Heterologous expression of this PKS in E. coli generated five new pharmaceutically valuable alkylresorcinols. Addition of glucose or pyruvate into the fermentation broths of E. coli expressing another type III PKS StTS resulted in a significant change in the product profiles. Five new products are produced and structurally characterized. Therefore, this work provides a new approach to generating new bioactive molecules in E. coli, the most widely used heterologous expression host.

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