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- All Subjects: High-throughput
- Creators: Caplan, Michael
- Member of: ASU Electronic Theses and Dissertations
- Resource Type: Text
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
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