Matching Items (3)
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- All Subjects: Metabolism
- All Subjects: Cryo-Electron-Microscopy
- Genre: Doctoral Dissertation
- Creators: Torres, Cesar
Description
Metabolic engineering is an extremely useful tool enabling the biosynthetic production of commodity chemicals (typically derived from petroleum) from renewable resources. In this work, a pathway for the biosynthesis of styrene (a plastics monomer) has been engineered in Escherichia coli from glucose by utilizing the pathway for the naturally occurring amino acid phenylalanine, the precursor to styrene. Styrene production was accomplished using an E. coli phenylalanine overproducer, E. coli NST74, and over-expression of PAL2 from Arabidopsis thaliana and FDC1 from Saccharomyces cerevisiae. The styrene pathway was then extended by just one enzyme to either (S)-styrene oxide (StyAB from Pseudomonas putida S12) or (R)-1,2-phenylethanediol (NahAaAbAcAd from Pseudomonas sp. NCIB 9816-4) which are both used in pharmaceutical production. Overall, these pathways suffered from limitations due to product toxicity as well as limited precursor availability. In an effort to overcome the toxicity threshold, the styrene pathway was transferred to a yeast host with a higher toxicity limit. First, Saccharomyces cerevisiae BY4741 was engineered to overproduce phenylalanine. Next, PAL2 (the only enzyme needed to complete the styrene pathway) was then expressed in the BY4741 phenylalanine overproducer. Further strain improvements included the deletion of the phenylpyruvate decarboxylase (ARO10) and expression of a feedback-resistant choristmate mutase (ARO4K229L). These works have successfully demonstrated the possibility of utilizing microorganisms as cellular factories for the production styrene, (S)-styrene oxide, and (R)-1,2-phenylethanediol.
ContributorsMcKenna, Rebekah (Author) / Nielsen, David R (Thesis advisor) / Torres, Cesar (Committee member) / Caplan, Michael (Committee member) / Jarboe, Laura (Committee member) / Haynes, Karmella (Committee member) / Arizona State University (Publisher)
Created2014
Description
This dissertation focuses on the biosynthetic production of aromatic fine chemicals in engineered Escherichia coli from renewable resources. The discussed metabolic pathways take advantage of key metabolites in the shikimic acid pathway, which is responsible for the production of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. For the first time, the renewable production of benzaldehyde and benzyl alcohol has been achieved in recombinant E. coli with a maximum titer of 114 mg/L of benzyl alcohol. Further strain development to knockout endogenous alcohol dehydrogenase has reduced the in vivo degradation of benzaldehyde by 9-fold, representing an improved host for the future production of benzaldehyde as a sole product. In addition, a novel alternative pathway for the production of protocatechuate (PCA) and catechol from the endogenous metabolite chorismate is demonstrated. Titers for PCA and catechol were achieved at 454 mg/L and 630 mg/L, respectively. To explore potential routes for improved aromatic product yields, an in silico model using elementary mode analysis was developed. From the model, stoichiometric optimums maximizing both product-to-substrate and biomass-to-substrate yields were discovered in a co-fed model using glycerol and D-xylose as the carbon substrates for the biosynthetic production of catechol. Overall, the work presented in this dissertation highlights contributions to the field of metabolic engineering through novel pathway design for the biosynthesis of industrially relevant aromatic fine chemicals and the use of in silico modelling to identify novel approaches to increasing aromatic product yields.
ContributorsPugh, Shawn (Author) / Nielsen, David (Thesis advisor) / Dai, Lenore (Committee member) / Torres, Cesar (Committee member) / Lind, Mary Laura (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2016
Description
The list of applications of plasmonic nanoparticles in the fields of energy research, sensing, and diagnostics and therapeutics is continuously growing. Processes for the synthesis of the nanoparticles for such applications should incorporate provision to easily functionalize the particle formed and should ideally not use toxic reagents or create toxic by-products. The traditional methods of synthesizing nanoparticles generally are energy inefficient, requires stringent conditions such as high temperature, pressure or extreme pH and often produces toxic by-products. Although there exist a few solution-based methods to solve this problem, there is one avenue which has recently gained attention for nanoparticle synthesis: using biomolecules to facilitate nanomaterials synthesis. Using biomolecules for synthesis can provide a template to guide the nucleation process and helps to keep conditions biocompatible while also combining the step of functionalization of the nanoparticle with its synthesis through the biomolecule itself. The dissertation focuses on studying the bio-templated synthesis of two such noble metal nanoparticle which have biomedical applications: gold and platinum. In chapter 2, Gold Nanoparticles (GNP), with long-term stability, were synthesized using Maltose Binding Protein (MBP) as templating agent. The site of gold interaction on MBP was identified by X-ray crystallography. A novel gold binding peptide, AT1 (YPFGGSGGSGM), was designed based on the orientation of the residues in the gold binding site, identified through crystallography. This designed peptide was also shown to have stabilized and affected the growth rate of GNP formation, in similar manner to MBP. Further in chapter 3, a nanosensor was formulated using a variation of this GNP-MBP system, to detect and measure ionizing radiation dose for cancer radiation therapy. Upon exposure to therapeutic levels of ionizing radiation, the MBP‐based sensor system formed gold nanoparticles with a dose‐dependent color that could be used to predict the amount of delivered X‐ray dose. In chapter 4, a similar system of protein templated synthesis was introduced for platinum nanoparticle (PtNP). Here, GroEL, a large homo-tetradecamer chaperone from E.coli, was used as templating and stabilizing agent for reduction of K2PtCl4 ions to form PtNP. To understand how GroEL interacts with the PtNPs and thereby stabilizes them, single-particle cryo-electron microscopy technique was used to model the complex in solution. A 3.8-Å resolution 3D cryo-EM map of GroEL depicting the location of PtNP inside its central cylindrical cavity was obtained. Fitting a GroEL model to the map revealed Arginine-268 from two adjacent subunits of GroEL interacting with the PtNP surface. Finally in chapter 5, a solution to the potential issues of single particle data processing on protein nanoparticle complexes, specifically with 2D classification, was developed by creating masking algorithms.
ContributorsThaker, Amar Nilkamal (Author) / Nannenga, Brent L (Thesis advisor) / Acharya, Abhinav (Committee member) / Torres, Cesar (Committee member) / Mills, Jeremy (Committee member) / Rege, Kaushal (Committee member) / Arizona State University (Publisher)
Created2020