title: Exploring the Capacity of Bacteria for Natural Product Biosynthesis
Creators: name: Fidan, Ozkan
Publisher: name: Utah State University
Year: 2019

description: This dissertation is focused on exploring the potential of bacteria for the biosynthesis of natural products with the purposes of generating novel natural product derivatives and of improving the titer of pharmaceutically important natural products.

A wide variety of compounds from various sources have been historically used in the treatment and prevention of diseases. Natural products as a major source of new drugs are extensively explored due to their huge structural diversity and promising biological activities such as antimicrobial, anticancer, antifungal, antiviral and antioxidant properties. For instance, penicillin as an early-discovered antimicrobial agent has saved millions of lives, indicating the historical importance of natural products. However, the alarming rise in the prevalence of drug resistance is a serious threat to public health and it has coincided with the decreasing supply of new antibiotics. Bacteria with a tremendous undiscovered potential have still been one of the richest sources of bioactive compounds to tackle the growing threat of antibiotic-resistant pathogens. Nevertheless, the production level of those important compounds is often quite low, and often undetectable using current analytical techniques. To expand the chemical repertoire of nature and to increase the titer of the natural products, researchers have developed various strategies, such as heterologous expression, co-cultivation of different bacteria, optimization of fermentation conditions, discovery of new species, engineering of biosynthetic enzymes, and manipulating regulatory elements. Thus, in my dissertation research, I have exploited a few of these strategies. First, I heterologously expressed some of the biosynthetic genes from the sch biosynthetic gene cluster, resulted in the production of a novel glycosylated angucycline. I was also able to generate another new glycosylated derivative of angucycline through gene disruption of tailoring enzymes. In this research, I isolated two novel angucycline derivatives and gained new insights into the glycosylation steps in the biosynthesis of Sch47554 and Sch47555. Next, I engineered the regulatory elements in Streptomyces sp. SCC-2136 through the overexpression and targeted gene disruption approaches for enhanced production of pharmaceutically important angucyclines. The highest titer of Sch47554 was achieved in Streptomyces sp. SCC-2136/ΔschA4 (27.94 mg/L), which is significantly higher than the wild type. This work thus provides an initial understanding of functional roles of regulatory elements in the biosynthesis of Sch47554 and Sch47555 and several engineered strains with enhanced production of Sch47554. Last, I isolated a carotenoid-producing endophytic bacterium from the leaves of the yew tree and optimized the fermentation conditions for an improved yield of zeaxanthin diglucoside up to 206 ± 6 mg/L. With the introduction of an additional copy of the Pscrt gene cluster through an expression plasmid, the engineered strain Pseudomonas sp. 102515/pOKF192 produced zeaxanthin diglucoside at 380 ± 12 mg/L, which is 85% higher than the parent strain. This strain holds a great potential for the production of pharmaceutically important antioxidant agent, zeaxanthin diglucoside.