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Title:Natural products discovery and characterization via synthetic biology
Author(s):Luo, Yunzi
Director of Research:Zhao, Huimin
Doctoral Committee Chair(s):Zhao, Huimin
Doctoral Committee Member(s):Lu, Yi; Kraft, Mary L.; Harley, Brendan A.
Department / Program:Chemical & Biomolecular Engr
Discipline:Chemical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Natural Products Discovery
Synthetic Biology
polycyclic tetra macrolactams (PTM) biosynthesis
Abstract:Microorganisms and plants have evolved to produce a myriad array of natural products that are of biomedical importance. Recent advances in synthetic biology have revolutionized our ability to discover and manipulate natural products biosynthetic pathways. This thesis describes in depth our efforts to enable natural products discovery and characterization via synthetic biology approaches. Type III PKSs produce a wide array of aromatic structures in spite of their structural simplicity. An efficient strategy for cloning and characterizing Type III PKS genes that are likely responsible for the synthesis of novel natural products from Eucalyptus species were developed. Five unique putative Type III PKSs genes were identified using this approach and two full-length genes were cloned and the biochemically characterized. With the recent development in genome sequencing projects, cryptic pathways serve as a potential source for novel natural products discovery. A synthetic biology approach for cryptic pathway activation and characterization was developed. One cryptic pathway from Streptomyces griseus, containing a PKS/NRPS hybrid gene, was chosen as a model system. To decipher this cluster, we have developed a plug-and-play platform based on the “DNA assembler” method. In this platform, one constitutive promoter was inserted in front of each gene involved in the pathway. qPCR data confirmed the increased transcription levels and HPLC data showed the formation of new polycyclic tetra macrolactams (PTMs) compounds. However, for PTM biosynthesis, no clear mechanism has been reported. Therefore, Chapter 4 describes the characterization of this PTM biosynthesis pathway by applying the same synthetic biology approach. We identified the boundary of this gene cluster by assembling a seven-gene construct and proposed a biosynthesis mechanism by studying a series of single-gene deletion and multiple-gene deletion constructs. Noticing that this one single gene cluster may have the potential to produce multiple products with closely related chemical structures, we biochemically characterized the modification enzymes in vitro and studied a phylogenetically related pathway as well. Finally, after achieving the success in the applications of our newly developed natural product discovery platform, we decided to make it more generally applicable for natural product discovery in actinomycetes. Additional strong constitutive promoters were identified via RNA-seq technique. The selected strong promoters were characterized based on both qPCR data and XylE enzyme specific activity assay. In total, 10 constitutive promoters were identified to be stronger than ermE*p, a widely used strong promoter reported in literature. These promoters will be used in our genomics-driven, synthetic biology platform for high throughput discovery of novel natural products in actinomycetes.
Issue Date:2014-05-30
Rights Information:Copyright 2014 Yunzi Luo
Date Available in IDEALS:2014-05-30
Date Deposited:2014-05

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