Chemical Engineering

Permanent URI for this collectionhttps://uwspace.uwaterloo.ca/handle/10012/9904

This is the collection for the University of Waterloo's Department of Chemical Engineering.

Research outputs are organized by type (eg. Master Thesis, Article, Conference Paper).

Waterloo faculty, students, and staff can contact us or visit the UWSpace guide to learn more about depositing their research.

Browse

Browsing Chemical Engineering by Subject "3-hydroxyvalerate"

Filter results by typing the first few letters
Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Integrated metabolic engineering and bioprocessing strategies for production of succinyl-CoA-derived chemicals in Escherichia coli
    (University of Waterloo, 2020-09-15) Miscevic, Dragan
    Due to various social, environmental, and technological issues associated with petro-based chemical processes, bio-based production using microbial cell factories has been recognized as a modern biotechnology for more renewable, sustainable, and clean manufacturing of chemical compounds. With respect to conventional chemical transformation, such in vivo biotransformation offers a substantial processing simplicity and technological leverage, particularly for the production of structurally complex compounds, by carrying out intricate and multi-step reactions with high specificity via one-pot reaction. Among numerous microbial cell factories, bacterium Escherichia coli is the most popular and user-friendly workhorse primarily due to its genetic amenability and well-developed bioprocessing strategies. While wild-type E. coli is not a native producer for many chemical products, technological advances in synthetic biology, genetic engineering, protein engineering, and metabolic engineering have offered a promise for extensive tailoring of E. coli strains with virtually any types of biosynthetic capacity. In this thesis study, we integrate several strain and bioprocess engineering strategies for rewiring of carbon flux around one of tightly-regulated central metabolic branch-points (i.e., node of succinyl-CoA) to enhance precursor supply for effective biosynthesis of three particular value-added chemicals, i.e., propionate, 3-hydroxyvalerate (3-HV), and 5-aminolevulinate (5-ALA), in E. coli host strains. In our initial study, we use our previously-derived propanologenic (i.e., 1-propanol-producing) bacterium E. coli strain with an activated genomic Sleeping beauty mutase (Sbm) operon for heterologous propionate production. Note that activated Sbm pathway branches out of the tricarboxylic acid (TCA) cycle at the succinyl-CoA node to form propionyl-CoA and its derived metabolites of 1-propanol and propionate. We first investigate the sensitivity around succinyl-CoA node by targeting multiple genes encoding enzymes that are involved in reactions contributing to carbon flux toward this node. These particular reactions are from three TCA cycle routes, i.e., oxidative TCA cycle, reductive TCA branch, and glyoxylate shunt. Effective blocking of oxidative TCA cycle and deregulating glyoxylate shunt has led to the secretion of roughly 30.9 g l-1 of propionate with minimal byproduct formation upon fed-batch cultivation our engineered strain under aerobic conditions. To best of our knowledge, the propionate titer reached in this study is the highest reported in E. coli from single structurally-unrelated carbon source (i.e., glycerol). For the subsequent part of our thesis, we further engineer our Sbm-activated E. coli strain by constructing a 3-hydroxyacid pathway for the production of heterologous long (odd)-chain 3-HV. The development of this particular strain involved modular construction of 3-HV biosynthetic pathway by establishing efficient Claisen condensation, reduction reaction, and CoA removing capabilities. In addition to strain engineering, various biochemical strategies (i.e., cultivation temperature, carbon sources, and aeration) were investigated for high secretion of approximately 3.71 g l-1 3-HV under vent-cap shake-flask cultivation. We used the information obtained from previous sections to scale-up our 3-HV production under batch and fed-batch bioreactor system. Herein, we used bioprocess engineering approach to enable more carbon flux toward succinyl-CoA node (and ultimately propionyl-CoA) by simultaneously inhibiting oxidative TCA cycle and deregulating glyoxylate shunt. With these developed strategies, we demonstrated effective cell growth, minimum secretion of byproducts, and high 3-HV production (up to 10.6 g l-1) in aerobic fed-batch culture. As far as we are aware, the level of 3-HV reached in this study is the highest reported for any microbial strain thus far. In our final study, we investigate the feasibility of our developed TCA cycle engineering strategy for effective production of 5-ALA, an endogenous non-proteinogenic amino acid. We first manipulated the non-native Shemin part of heme biosynthetic pathway by downregulating essential 5-ALA-consuming reaction using Clustered Regularly Interspersed Short Palindromic Repeats interference (CRISPRi), thus considerably preserving intracellular pool of this target metabolite. As Shemin pathway synthesized 5-ALA in a one-step reaction catalyzed by molecular fusion of succinyl-CoA and glycine, formation of 5-ALA was further improved by channeling more carbon toward succinyl-CoA precursor in TCA cycle, reaching high titer of 6.93 g l-1 under aerobiosis. To best of our knowledge, the consolidated strain and bioprocess engineering strategy reported in this study represents one of the most effective and economical bio-based production of 5-ALA in E. coli from single carbon source. Collectively, this thesis attempts to highlight the importance and applicability of integrated strain engineering and bioprocessing strategies for enhanced bio-based production of industrially valuable products. Harnessing this enhanced carbon flux toward succinyl-CoA node and employing various modular chain elongation and pathway enzymes can open the avenue for the controlled production of various other valuable succinyl-CoA-derived chemicals.
  • Thumbnail Image
    Item
    Production of 3-hydroxy acids from acetate in engineered Escherichia coli
    (University of Waterloo, 2018-09-21) Mozell, Bradley
    Acetate serves as an uncommon carbon source for metabolic engineering of value-added chemicals. As a carbon source, acetate has unique advantages since it is not derived from food sources and can be produced as a waste by-product from many biological processes. This allows acetate to be an inexpensive feed source for biomanufacturing whose use would not affect food prices. However, use of acetate as a feed source for bioproduction is limited by its inhibitory effects. As such, fine chemicals, which have high value and low production demand, are preferred target products for bioproduction using acetate feed. 3-hydroxy acids, i.e. 3-hydroxybutyrate (3-HB) and 3-hydroxyvalerate (3-HV), are one such example of fine chemicals. This study demonstrates the production of 3-HB and 3-HV in recombinant Escherichia coli, using acetate as a sole carbon source. Various metabolic pathways related to acetate metabolism such as; acetate assimilation, glyoxylate shunt, glycolytic and gluconeogenic pathways related to the phosphoenolpyruvate-pyruvate node, as well as global regulatory systems, were manipulated through targeted gene knockouts, to boost the production of 3-hydroxy acids. These genetic manipulations of the base 3-hydroxy acid-producing strain of E. coli resulted in the production of up to 1.75 g/L of 3-HB and 0.571 g/L of 3-HV (individually).