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- All Subjects: chemical engineering
- All Subjects: Metabolism
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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
The effects of specific histone deacetylase inhibitors (HDACi) on transgene expression in combination with a novel polymer as a delivery vehicle are investigated in this research. Polymer vectors, although safer than viruses, are notorious for low levels of gene expression. In this investigation, the use of an emerging chemotherapeutic anti-cancer drug molecule, HDACi, was used to enhance the polymer-mediated gene expression. HDACi are capable of inhibiting deacetylation activities of histones and other non-histone proteins in the cytoplasm and nucleus, as well as increase transcriptional activities necessary for gene expression. In a prior study, a parallel synthesis and screening of polymers yielded a lead cationic polymer with high DNA-binding properties, and even more attractive, high transgene expressions. Previous studies showed the use of this polymer in conjunction with cytoplasmic HDACi significantly enhanced gene expression in PC3-PSMA prostate cancer cells. This led to the basis for the investigation presented in this thesis, but to use nuclear HDACi to potentially achieve similar results. The HDACi, HDACi_A, was a previously discovered lead drug that had potential to significantly enhance luciferase expression in PC3-PSMA cells. The results of this study found that the 20:1 polymer:plasmid DNA weight ratio was effective with 1 uM and 2 uM HDACI_A concentrations, showing up to a 9-fold enhancement. This enhancement suggested that HDACi_A was effectively aiding transfection. While not an astounding enhancement, it is still interesting enough to investigate further. Cell viabilities need to be determined to supplement the results.
ContributorsLehrman, Jennifer (Author) / Rege, Kaushal (Thesis advisor) / Caplan, Michael (Committee member) / Pizziconi, Vincent (Committee member) / Arizona State University (Publisher)
Created2012
Description
The diversity of industrially important chemicals that can be produced biocatalytically from renewable resources continues to expand with the aid of metabolic and pathway engineering. In addition to biofuels, these chemicals also include a number of monomers with utility in conventional and novel plastic materials production. Monomers used for polyamide production are no exception, as evidenced by the recent engineering of microbial biocatalysts to produce cadaverine, putrescine, and succinate. In this thesis the repertoire and depth of these renewable polyamide precursors is expanded upon through the engineering of a novel pathway that enables Escherichia coli to produce, as individual products, both δ-aminovaleric acid (AMV) and glutaric acid when grown in glucose mineral salt medium. δ-Aminovaleric acid is the monomeric subunit of nylon-5 homopolymer, whereas glutaric acid is a dicarboxylic acid used to produce copolymers such as nylon-5,5. These feats were achieved by increasing endogenous production of the required pathway precursor, L-lysine. E. coli was engineered for L-lysine over-production through the introduction and expression of metabolically deregulated pathway genes, namely aspartate kinase III and dihydrodipicolinate synthase, encoded by the feedback resistant mutants lysCfbr and dapAfbr, respectively. After deleting a natural L-lysine decarboxylase, up to 1.6 g/L L-lysine could be produced from glucose in shake flasks as a result. The natural L-lysine degradation pathway of numerous Pseudomonas sp., which passes from L-lysine through both δ-aminovaleric acid and glutaric acid, was then functionally reconstructed in a piecewise manner in the E. coli L-lysine over-producer. Expression of davBA alone resulted in the production of over 0.86 g/L AMV in 48 h. Expression of davBADT resulted in the production of over 0.82 g/L glutaric acid under the same conditions. These production titers were achieved with yields of 69.5 and 68.4 mmol/mol of AMV and glutarate, respectively. Future improvements to the ability to synthesize both products will likely come from the ability to eliminate cadaverine by-product formation through the deletion of cadA and ldcC, genes involved in E. coli's native lysine degradation pathway. Nevertheless, through metabolic and pathway engineering, it is now possible produce the polyamide monomers of δ-aminovaleric acid and glutaric acid from renewable resources.
ContributorsAdkins, Jake M (Author) / Nielsen, David R. (Thesis advisor) / Caplan, Michael (Committee member) / Torres, Cesar (Committee member) / Arizona State University (Publisher)
Created2012
Description
Early humans adapted to eating cooked food with increased energy density and absorption of macronutrients. However, in modern times many suffer from diseases like obesity and type 2 diabetes which can result from too much energy being absorbed from food. This study measures glucose responses to a high glycemic meal with a side dish of raw or cooked vegetables. There was a slight trend for raw vegetables to have decreased postprandial blood glucose responses when compared to cooked vegetables.
ContributorsWilkins, Christine Marie (Author) / Johnston, Carol (Thesis director) / Jacobs, Mark (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor)
Created2014-05
Description
Relapse after tumor dormancy is one of the leading causes of cancer recurrence that ultimately leads to patient mortality. Upon relapse, cancer manifests as metastases that are linked to almost 90% cancer related deaths. Capture of the dormant and relapsed tumor phenotypes in high-throughput will allow for rapid targeted drug discovery, development and validation. Ablation of dormant cancer will not only completely remove the cancer disease, but also will prevent any future recurrence. A novel hydrogel, Amikagel, was developed by crosslinking of aminoglycoside amikacin with a polyethylene glycol crosslinker. Aminoglycosides contain abundant amount of easily conjugable groups such as amino and hydroxyl moieties that were crosslinked to generate the hydrogel. Cancer cells formed 3D spheroidal structures that underwent near complete dormancy on Amikagel high-throughput drug discovery platform. Due to their dormant status, conventional anticancer drugs such as mitoxantrone and docetaxel that target the actively dividing tumor phenotype were found to be ineffective. Hypothesis driven rational drug discovery approaches were used to identify novel pathways that could sensitize dormant cancer cells to death. Strategies were used to further accelerate the dormant cancer cell death to save time required for the therapeutic outcome.
Amikagel’s properties were chemo-mechanically tunable and directly impacted the outcome of tumor dormancy or relapse. Exposure of dormant spheroids to weakly stiff and adhesive formulation of Amikagel resulted in significant relapse, mimicking the response to changes in extracellular matrix around dormant tumors. Relapsed cells showed significant differences in their metastatic potential compared to the cells that remained dormant after the induction of relapse. Further, the dissertation discusses the use of Amikagels as novel pDNA binding resins in microbead and monolithic formats for potential use in chromatographic purifications. High abundance of amino groups allowed their utilization as novel anion-exchange pDNA binding resins. This dissertation discusses Amikagel formulations for pDNA binding, metastatic cancer cell separation and novel drug discovery against tumor dormancy and relapse.
Amikagel’s properties were chemo-mechanically tunable and directly impacted the outcome of tumor dormancy or relapse. Exposure of dormant spheroids to weakly stiff and adhesive formulation of Amikagel resulted in significant relapse, mimicking the response to changes in extracellular matrix around dormant tumors. Relapsed cells showed significant differences in their metastatic potential compared to the cells that remained dormant after the induction of relapse. Further, the dissertation discusses the use of Amikagels as novel pDNA binding resins in microbead and monolithic formats for potential use in chromatographic purifications. High abundance of amino groups allowed their utilization as novel anion-exchange pDNA binding resins. This dissertation discusses Amikagel formulations for pDNA binding, metastatic cancer cell separation and novel drug discovery against tumor dormancy and relapse.
ContributorsGrandhi, Taraka Sai Pavan (Author) / Rege, Kaushal (Thesis advisor) / Meldrum, Deirdre R (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Caplan, Michael (Committee member) / Tian, Yanqing (Committee member) / Arizona State University (Publisher)
Created2016