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Title:Pathway engineering for the discovery and optimized production of phosphonic acids
Author(s):Freestone, Todd S
Director of Research:Zhao, Huimin
Doctoral Committee Chair(s):Zhao, Huimin
Doctoral Committee Member(s):Leckband, Deborah E.; Metcalf, William W.; Rao, Christopher V.
Department / Program:Chemical & Biomolecular Engr
Discipline:Chemical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Phosphonic acids
metabolic engineering
pathway refactoring
Abstract:Natural products have been a great benefit to mankind, especially in modern times. With approximately half of all drugs used today being derived from small molecules observed in nature, the impact of these compounds is immeasurable. In the mid-twentieth century, a period known as the Golden Age of Antibacterials, the natural product field experienced a wave of discovery that has yet to be replicated. Even with the increased focus on using synthetic chemistry to discover potential pharmaceuticals, there has been a steady decline in discovery rates over the past decades. While traditional natural product discovery methods are limited to what is detectable in nature, advances in DNA sequencing technology, bioinformatics, and molecular biology have given researchers the ability to mine genomes for new compounds. By combing the abundant wealth of available genomic data, biosynthetic gene clusters can be readily identified for exploitation. However, two bottlenecks impede the transition from identified gene cluster to useful drug. The first hurdle is identifying the molecule linked to a set of biosynthetic genes. This step is well beyond any computational approach and must rely on empirical substantiation. The second hurdle is to produce enough of the compound in an economically feasible manner. In this work we implement pathway engineering strategies to further lower these barriers, with a focus on the class of natural products called phosphonic acids. These compounds have a stable carbon-phosphorus bond, which allow them to mimic phosphate esters and carboxylic acids, making them potential enzyme inhibitors. For the antimalarial phosphonate FR900098, a novel pathway engineering strategy, called enriched library screening, was developed that allows one to home in on top pathways in combinatorial libraries. When applied to the FR900098 pathway, a strain with a significant increase in production was found. This method can also be applied to other natural product pathways to rapidly find expression configurations that give higher yields. Additional strain engineering was also undertaken on the FR900098 strain; however, improvements were not seen despite a number of gene knockouts and knockdowns tested. To discover new phosphonic acids, biosynthetic pathways from two actinobacteria, Streptomyces species NRRL F-525 and Kibdelosporangium aridum largum NRRL B-24462, were fully engineered for production in the expression host Streptomyces lividans. This was done by placing the individual biosynthetic genes downstream of promoters characterized in S. lividans. Phosphonate production was seen in both hosts, with a novel phosphonate being identified in the Streptomyces species NRRL F-525 cluster. This approach can also be extended to discover other natural product gene clusters.
Issue Date:2016-12-02
Rights Information:Copyright 2016 Todd Steven Freestone
Date Available in IDEALS:2017-03-01
Date Deposited:2016-12

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