Date of Award:

5-2016

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biological Engineering

Committee Chair(s)

Jixun Zhan

Committee

Jixun Zhan

Committee

Dong Chen

Committee

Foster Agblevor

Committee

Randy Lewis

Committee

David W. Britt

Abstract

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

Plant natural products are an important source of therapeutics, such as paclitaxel (anticancer) and artemisinin (anti-malarial). Production of plant natural products relies on conventional plant cultivation and solvent extraction, which is time-consuming and cost-ineffective. This work built a biosynthetic platform in Escherichia coli using seven biosynthetic genes from plants and bacteria, which were used to make valuable compounds such as the strong antioxidant resveratrol and anti-inflammatory agent curcuminoids. Through different combinations of these genes, E. coli was engineered to produce four phenylpropanoid acids (cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid), three bioactive natural stilbenoids (resveratrol, piceatannol and pinosylvin), and three natural curcuminoids (curcumin, bisdemethoxycurcumin and dicinnamoylmethane). “Unnatural” natural products including dicafferolmethane and 2-chloro-resveratrol were also generated by modifying the existing pathways. Based on the color of curcuminoids, a novel and efficient visible reporter assay was established for screening of phenylalanine ammonia-lyase (PAL), an enzyme involved in the first biosynthetic step of many plant natural products.

Actinomycetes are a group of bacteria well-known for the production of antibiotics. A strong antibacterial agent, dutomycin, was discovered from Streptomyces minoensis NRRL B-5482. Through genome sequencing and targeted gene disruption, a type II polyketide biosynthetic gene cluster was found to be responsible for the assembly of dutomycin. Several key enzymes in the pathway were functionally characterized and a series of new analogs were generated, including a new compound with more promising antibacterial activity.

Metabolic engineering of natural product biosynthetic pathways showed its promise for creating and producing valuable compounds with chemical diversity for drug discovery. This research has biosynthesized 17 valuable molecules with medicinally relevant bioactivities. The results from this study provided important platforms and technical basis for further engineering polyketide biosynthetic pathways to produce valuable bioactive molecules and generate novel analogs for bioactivity screening and drug development.

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