Browsing by Subject "Metabolic engineering"

Now showing 1 - 6 of 6
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    Biosynthetic routes to short-chain carboxylic acids and their hydroxy derivatives
    (2013-07) Dhande, Yogesh Khemchandra
    The current drive for sustainable routes to industrial chemicals and energy due to depleting fossil reserves has motivated research in the field of biotechnology. Biomass is abundant on earth and photosynthesis, if utilized properly, can lead the way to a sustainable carbon cycle. The recent advances in metabolic engineering, DNA sequencing, and protein engineering are driving research to achieve this goal. In this work, the native leucine and isoleucine biosynthetic pathways in Escherichia coli were expanded for the synthesis of pentanoic acid (PA) and 2-methylbutyric acid (2MB) respectively. Several aldehyde dehydrogenases and 2-ketoacid decarboxylases were studied for the conversion of ketoacids into respective carboxylic acids. The optimal combinations of these enzymes enabled production of 2.6 g/L 2MB with IPDC-AldH and 2.6 g/L of PA with IPDC-KDHba in shake-flask fermentations. The extension of these pathways to synthesize value-added chemicals like hydroxyacids was attempted. The cytochrome P450 BM-3 enzyme was engineered to enhance its ability to oxidize unactivated C-H bonds in the short-chain carboxylic acids. Nine mutants were created by rational. The mutant L437K showed 20 to 30-fold higher activity compared to the wild-type. A screening strategy was developed to evolve isobutyrate-hydroxylating activity in P450s based on the valine degradation pathway in Pseudomonas aeruginosa. As a growth-based selection strategy, it will allow screening of a large library. This work expands the library of chemicals that can be produced biologically as a step forward to the sustainable future.
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    Discovery and characterization of sesquiterpenoid biosynthetic pathways from Basidiomycota
    (2014-01) Wawrzyn, Grayson Thomas
    Terpenoids are the largest group of secondary metabolites and can be found in organisms ranging from microscopic bacteria to large plant species. These compounds are commonly used for signaling and defense against ecological predators/competitors and possess many unique and interesting biological activities. Many compounds possess enough value that large scale production of terpenoids for biofuel (e.g. farnesene) and pharmaceutical (e.g. artemisinin) applications are well underway. The goal of this work was and still is to uncover the biological machinery responsible for making the bioactive terpenoid compounds. This requires a deep understanding of the underlying biochemistry as well as the genetic organization of the organism from which you are trying to extract said machinery. In the context of this research the biosynthetic machinery is the enzymes (primarily sesquiterpene synthases and P450 monooxygenases) responsible for synthesizing the anticancer illudin terpenoids, and the target organism is the fungus Omphalotus olearius. To accomplish this goal I identified the first biosynthetic pathway enzyme, a protoilludene synthase, and identified putative modifying enzymes that catalyze further modification of the first pathway intermediate. Arguably more importantly, I chose to use our biochemical data to better understand sesquiterpene synthases across many fungal genera. This work provides significant breakthroughs in understanding illudin biosynthesis and also provides a predictive framework for further examination of terpenoid biosynthesis in many different fungal species.
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    Elementary mode analysis of Ralstonia Eutropha H16 metabolism for the production of useful metabolites
    (2013-05) Lopez, Gilsinia
    Ralstonia eutropha H16 is a Gram-negative, facultative chemolithoautotrophic bacterium with the capability to synthesize many useful metabolites. One of the keys to the organism's lifestyle is its ability to use--alternatively or concomitantly--both organic compounds and molecular H2 as sources of energy. It can fix CO2 via the Calvin-Benson-Bassham (CBB) cycle and produces several useful metabolites like Poly(3-hydroxybutyric acid), isobutanol, 2,3-butanediol and ethanol. To quantitatively evaluate the capabilities of the metabolism we have set up models of the central metabolism that is based on known pathways of the organism. The lithoautotrophic metabolic model consists of 29 reversible reactions, 33 irreversible reactions, 59 internal metabolites and 11 external metabolites exchanged through the cell membrane. The heterotrophic model with fructose as a substrate has 31 reversible reactions, 47 irreversible reactions, 66 internal metabolites and 13 external metabolites. Elementary Mode Analysis identified 759 modes during lithoautotrophic growth and 135074 modes during heterotrophic growth. We have used the results from this analysis to predict key genetic alterations in the metabolism that would direct the metabolic flux towards the production of ethanol, isobutanol or polyhydroxybutanoate.
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    Genomic Analysis And Engineering Of Chinese Hamster Ovary Cells For Improved Therapeutic Protein Production
    (2020-05) O'Brien, Sofie
    Protein biologics have transformed the field of medicine in recent years. These complex molecules are produced in living cells, primarily Chinese Hamster Ovary (CHO) cells. Due to the importance of these therapeutic proteins to disease treatment, it is essential to improve the efficiency of their production, both to promote the development of new therapies, and to bring down the cost of manufacturing. One of the most important components of the production process is the development of a cell line. Many features of a cell line, such as cellular growth, metabolism, and the integration site of the gene encoding the protein, influence the resulting culture productivity and quality of the protein produced. In this thesis, multiple aspects of the relationship between integration site and resulting cell line behavior were investigated. First, a rapid integration site identification method was developed to facilitate further analysis of integration sites in complex cell lines. Next, to examine genomic instability, parental cells were compared with high and low producing subclones, leading to identification of genomic regions vulnerable to copy gain/loss. A large-scale analysis across many CHO cell lines was further performed to look for global regions of genomic variation, independent of an individual cell line. To evaluate integration sites with high transcriptional potential, integration sites from high producing cells were examined, and high transgene expression was correlated to high transcriptional activity and accessibility of the integration region. This work also extended to energy metabolism, another key feature of a cell line. Through the use of model guided multi-gene engineering to manipulate cell metabolism, waste product generation was reduced in late-stage culture. With these tools and technologies, we can build a more complete picture of a desirable integration site, which can be used to drive the development of next generation cell lines with high, stable expression of transgenes for therapeutic protein production.
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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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    Synthetic Biology Approach to New Sustainable Materials
    (2018-03) McClintock, Maria
    Rapid industrialization and an abundance of cheap petroleum fueled the production and development of a great variety of synthetic polymers in the twentieth century. Over the past 60 years, these materials have become a part of the fabric of modern life. They are pervasive; from the polyurethanes in our cars and furniture, to the polypropylene in a state of the art medical implant, we rely on synthetic polymers every day of our lives, to accomplish tasks both trivial and critical. However, production of these chemicals from petroleum feedstocks is unsustainable and damaging to the environment. One potential option for more sustainable production is to use microbial fermentation to generate industrial chemicals. Microbial fermentation offers the opportunity to produce chemicals from biomass, making the compounds produced renewable feedstocks. Furthermore, the conditions used for, and byproducts produced from, microbial fermentation are benign. However, many microbial monomers face challenges in terms of economic viability and utility. With this in mind, my PhD research has focused on developing engineering systems for production of novel and viable monomers, as well as implementation of biological monomers for material applications. Using metabolic engineering, I have implemented the first heterologous pathway for production of dipicolinic acid, an aromatic di-acid that could be used as a biological replacement for isophthalic acid, a major component of the performance polymers Nomex® and polybenzimidazole, as well as a useful additive in many other polymers. By working with collaborators, I have used ancestral reconstruction to improve the production of anhydromevalonolactone, a monomer that can serve as a sustainable alternative to poly(acrylate). Finally, I have worked to establish a new platform for developing zwitterionic materials. In this project, we were able to engineer E. coli to produce N-acetyl-serine, a compound that can be dehydrated to form an acrylate monomer with a protected amine. I then polymerized this monomer with styrene and developed zwitterionic coatings that show improved resistance to cell adhesion. Overall, my work has contributed to the development of new metabolic pathways and material applications of biologically-derived monomers.