Date of Award:

8-2023

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

Yu Huang

Committee

Elizabeth Vargis

Committee

Cheng-Wei Tom Chang

Abstract

This dissertation is mainly focused on exploring versatile glycosyltransferases from different microorganisms with the purpose of biosynthesizing bioactive natural product glycosides through microbial fermentation. This engineered biosynthesis approach is environmentally friendly, safe, and efficient compared with plant extraction, chemical synthesis, and enzymatic synthesis. Additionally, we applied these characterized glycosyltransferases as efficient biocatalytic tools to produce novel glycosides to expand the chemical repertoire of nature. These novel compounds showed improved water solubility and some of them exhibited enhanced stability and biological activity, which laid the foundation for the development of new therapeutic agents.

In my dissertation research, I have discovered two versatile glycosyltransferases and utilized them to create novel glycosides of selected bioactive molecules. Firstly, I found that Beauveria bassiana ATCC 7159 and Streptomyces chromofuscus ATCC 49982 can glycosylate the anticancer agent 2'-hydroxyflavanone and antitubercular agent chlorflavonin into their glycosides, respectively. These two putative glycosyltransferases are both versatile enzymes. Specifically, B. bassiana ATCC 7159 can attach 4''-O-methyl-β-D-glucose to different hydroxy groups of 2'-hydroxyflavanone. Meanwhile, S. chromofuscus ATCC 49982 can append glucuronic acid to the 5-hydroxy group of chlorflavonin. Second, a highly flexible glucosyltransferase (BbGT) was identified from B. bassiana ATCC 7159. When purified to near homogeneity, BbGT can convert quercetin to five mono- and one di-glucosylated derivatives through an in vitro enzymatic reaction. Moreover, different quercetin glucosides, namely quercetin-7-O-β-D-glucopyranoside and quercetin-3-O-β-D-glucopyranoside, were respectively produced in engineered Escherichia coli and Saccharomyces cerevisiae harboring BbGT. A novel versatile microbial UDP-glucuronyltransferase (UGT) was then discovered and characterized from S. chromofuscus ATCC 49982. The UGT was expressed in E. coli and was capable of converting resveratrol into two glucuronides both in vitro and in vivo. Additionally, the UGT has a highly flexible substrate specificity and serves as an effective tool to prepare mono- or diglucuronides of various bioactive molecules. These two studies provide effective methods to produce valuable health-benefitting natural product glycosides by expressing these two versatile glycosyltransferases in different expression hosts. Finally, I used the two aforementioned biocatalytic tools together with other wild type strains to generate a series of novel glycosides of 2'-hydroxyflavone with improved water solubility and some of them displayed enhanced bioactivity. Thus, this dissertation research provides several effective biocatalysts that can be used for structural modification of polyphenolic compounds for enhanced water solubility and/or biological activities.

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