Browsing by Subject "Biosynthesis"

Now showing 1 - 5 of 5
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    Addressing unanswered questions in bacterial hydrocarbon biosynthesis
    (2017-10) Jensen, Matthew
    Modern society relies heavily on hydrocarbons. Because they are used as liquid transportation fuels, cosmetics, waxes, and food coatings, hydrocarbons are important components of most aspects of daily life. The majority of hydrocarbon products are extracted or derived from cude oil. Energy costs, fuel demand, and environmental concerns involving the non-renewable nature of petroleum-derived compounds have sparked recent interest in microbial hydrocarbon production. Engineering the diversity of microbial metabolic pathways to produce biofuels and chemicals represents a renewable alternative to fossil fuels. One pathway of interest is bacterial long-chain olefin biosynthesis. Divergent bacterial species have been shown to synthesize these waxy hydrocarbons using four enzymes: OleABCD. Recent investigations have aimed to understand how these enzymes work in concert to produce valuable hydrocarbon intermediates and products. These findings will be useful for future pathway engineering for renewable, bacterial olefin production. The first three chapters of this dissertation deal with elucidating the catalytic mechanism of the first enzyme in the olefin biosynthesis pathway, OleA. Chapters 2, 3, and 4 each investigate a separate amino acid necessary for OleA β-keto acid formation using methods of site-directed mutagenesis, biochemistry, and X-ray crystallography. In Chapter 2, the unique substrate binding channel architecture of OleA is directly demonstrated by trapping substrates and intermediates within Cys143 mutated enzymes. The role of Glu117 as the catalytic base needed to prime condensation through deprotonation of the second acyl-CoA substrate is established in Chapter 3. This represents the first dimeric thiolase superfamily enzyme that uses an active site base donated from the second monomer. It also provides evidence for the unique mechanistic strategy of OleA compared to other thiolases. Chapter 4 investigates the role His285 plays in positioning substrate and intermediates for productive condensation by OleA. It is also shown that His285 plays a role in protecting the Cys143 thiolate from oxidative damage. The dissertation concludes with the investigation of the catalytic function of OleC and the characterization of the multienzyme assembly formed by OleB, OleC, and OleD. In Chapter 6, OleC is demonstrated to produce β-lactones from β-hydroxy acids. This is the first example of a β-lactone synthetase, a novel enzyme function. It is also shown that OleC is homologous to amino acid sequences encoded in known β-lactone-producing natural product pathways, suggesting a common mechanism for β-lactone formation. Chapter 7 details the formation of an assembly consisting of OleBCD. Following co-expression of OleABCD, OleBCD are found to co-elute over nickel-affinity, anti-FLAG, and size-exclusion chromatographic purifications. These assemblies form ~2 MDa structures that produce cis-olefin following the addition of OleA and acyl-CoA. Negative stain transmission electron microscopy reveals a mixture of assemblies ranging from 24-40 nm in diameter. It is proposed that these assemblies are necessary for protecting the cell from the highly-reactive β-lactone intermediate.
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    Plant phenylpropanoid biosynthesis in Escherichia coli: engineering novel pathways and tools
    (2014-10) Bloch, Sarah E.
    Plant phenylpropanoid natural products are important in the discovery of safe and effective therapeutics. Most plant natural products cannot be economically mass produced via extraction from plant tissue or chemical synthesis. In recent decades, engineering microbes to carry out the biosynthesis of plant natural products has emerged as a powerful technology. The goal of this thesis was to expand the capabilities of microbial biosynthesis of plant phenylpropanoids in Escherichia coli through exploring novel biosynthetic pathways and metabolic engineering tools. I first explored the biosynthesis of valuable lignans in E. coli, establishing random oxidative radical coupling through overexpression of a laccase and attempting to show stereoselective coupling by a dirigent protein. I also designed and built a biosynthetic pathway for rosmarinic acid, a valuable hydroxycinnamic acid ester, showed pathway bottlenecks and limitations, and identified future optimization strategies. I have also begun a project to better understand cargo protein encapsulation within bacterial microcompartments and to develop their utility as a means of spatially organizing metabolic pathways. This work has contributed significantly to the field of microbial metabolic engineering and has laid the groundwork for future economically viable production platforms.
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    Sustainable Development of Polymers Using Hybrid Process of Biosynthesis and Chemical Reactions
    (2022-02) Wu, Yuxiao
    Natural and synthetic polymers can be found everywhere in our everyday lives. Polymer materials contribute to crucial roles in sophisticated applications such as electronics, medical devices and implants. They are also found in parts of every trivial thing in modern life such as clothing, food packaging, housing, and transportation. We rely more on synthetic polymers over natural polymers since the prosperity of petrochemical industry after the Second Industrial Revolution. The cheap and abundant fossil fuel allowed the development of a wide array of synthetic polymers in the 20th century. However, environmental concerns associated with using petroleum feedstock as the raw material received more and more attention. Over the past several decades researchers have focused on replacing petroleum feedstock with renewable feedstock to produce sustainable polymers. The abundant biomass is often used as the renewable raw material and one important way to breakdown biomass is through microbial fermentation. The development of genetic engineering allowed successful expansion of the natural metabolic pathways of microorganisms. Many industrial chemicals and novel chemicals were produced from microbial fermentation. The advantage of microbial fermentation is that it is carried out in mild reaction conditions and generates environmentally benign byproducts. However, microbial fermentation lacks the economic viability and utility due to long fermentation time and productivity. A hybrid synthesis process combining microbial fermentation and chemical reaction is a highly efficient approach to produce sustainable polymers. With this in mind, my PhD research has focused on developing hybrid chemical engineering process for the production of novel or existing polymer precursors and implement them for material applications. Using metabolic engineering and simple chemical reaction, I have produced N-acetyldopamine which can be used as a precursor for catechol functionalized polymers. By optimizing the fermentation pathway of mesaconic acid, the yield and productivity have reached an industrially practical level. The hybrid synthesis process to isoprene, the monomer for natural rubber, was established combining this optimized fermentation route with preciously developed one-pot cascade thermal chemical reaction. Finally, I implemented a heterologous pathway to produce citramalic acid. I have worked to establish a C5 diacid platform from microbial fermentation. Combining with a thermocatalytic decarboxylation reaction, an integrated hybrid process for the conversion of glucose to poly(methyl methacrylate) was achieved.
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    A unified strategy for penostatin (Bio)synthesis and forays in computational chemistry
    (2012-07) Jansma, Matthew James
    PART 1: Comprising Chapters I–IV, the studies described in the first part of this Thesis have as their overarching goal the utilization of organic synthesis to address questions of biosynthetic import. The impetus for this work has been provided by the penostatins A–I, a family of biologically active, structurally atypical, polyketide-derived secondary metabolites isolated from the fungus Penicillium sp. OUPS-79. Critical mechanistic and structural analyses have compelled the hypothesis that the penostatins arise via spontaneous (i.e., non enzyme-catalyzed) pericyclic reaction cascades that emanate from a single biogenetic precursor. Part 1 is inaugurated with a concise summary of the isolation, structure determination, and biological activity of the penostatins (Chapter I). In addition, a discussion of others' previous synthetic efforts toward members of the family is presented. Chapter II describes a campaign that has culminated in the stereoselective synthesis and study of the putative biosynthetic precursor to penostatins A and B, which constitutes the vast majority of the work conducted during the author's tenure. A pertinent model study that involved the design, synthesis, and subsequent investigation of (the enolates derived from) a pair of model dihydropyran substrates is detailed in Chapter III. This work has tentatively supported the notion that penostatins I and F arise via spontaneous [3,3]-sigmatropic (Claisen) rearrangements. Finally, Chapter IV documents progress toward the synthesis of a model substrate relevant to the biosynthesis of penostatins G and H. PART 2: The ability to reliably deduce the constitution and relative configuration of newly isolated organic molecules lies at the very core of all endeavors in the fields of synthetic organic and natural products chemistry. Nuclear magnetic resonance (NMR) spectroscopy is unarguably the single most powerful spectroscopic tool for this task; however, the unambiguous assignment of these structural properties via spectroscopic data alone is rarely a trivial matter. In Part 2 of this Thesis, the power and utility of DFT-based computational methods for the structure determination of small organic molecules are showcased. Chapter V includes a short discussion of the motivation for these studies and previous work from the Hoye/Cramer team. Then, in Chapter VI, computed proton (1H) and carbon (13C) NMR chemical shifts (δ) are employed to address structural issues that have arisen from two concurrent synthetic endeavors in the Hoye group. On the basis of the computational results described therein, reassignments of (i) the structures of the ‘Jones isomers’ and (ii) the relative configuration within (at least) the AB ring system of phomopsichalasin are strongly recommended. Additionally, a reexamination of the reported 1H NMR chemical shift assignments for patchouli alcohol has emerged from a collaborative effort with the Cramer group.
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    The Utilization of Algicidal Bacteria for Improved Lipid Extractions and Insights into Neutral Lipid Production in a Wax Ester Accumulating Bacterium
    (2013-08) Lenneman, Eric M.
    Based on finite and diminishing quantities of available fossil fuels and the increased demand by a growing population, the identification and production of biologically derived fuels and alternatives to petroleum based compounds has become increasingly important. Biological fuels hold promise from both an economical and environmental standpoint. Microorganisms may hold the key to producing these compounds as many algae and bacteria have been found to produce high quantities of lipids and other bioproducts similar to those obtained from fossil fuels. The purpose of these studies was to evaluate the potential application of algae-degrading bacteria to aid in lipid extractions from the microalgae Neochloris oleoabundans and Dunaliella tertiolecta. Separate studies evaluated the wax ester biosynthetic pathway in the lipid accumulating bacterium Marinobacter aquaeolei VT8 through the analysis of transcriptional levels within wild-type cells, and finally these studies were complimented by gene deletion efforts for specific enzymes within this biosynthetic pathway.