Matching Items (5)
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- All Subjects: Metabolism
- Creators: Katsanos, Christos
- 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
Skeletal muscle (SM) mitochondria generate the majority of adenosine triphosphate (ATP) in SM, and help regulate whole-body energy expenditure. Obesity is associated with alterations in SM mitochondria, which are unique with respect to their arrangement within cells; some mitochondria are located directly beneath the sarcolemma (i.e., subsarcolemmal (SS) mitochondria), while other are nested between the myofibrils (i.e., intermyofibrillar (IMF) mitochondria). Functional and proteome differences specific to SS versus IMF mitochondria in obese individuals may contribute to reduced capacity for muscle ATP production seen in obesity. The overall goals of this work were to (1) isolate functional muscle SS and IMF mitochondria from lean and obese individuals, (2) assess enzyme activities associated with the electron transport chain and ATP production, (3) determine if elevated plasma amino acids enhance SS and IMF mitochondrial respiration and ATP production rates in SM of obese humans, and (4) determine differences in mitochondrial proteome regulating energy metabolism and key biological processes associated with SS and IMF mitochondria between lean and obese humans.
Polarography was used to determine functional differences in isolated SS and IMF mitochondria between lean (37 ± 3 yrs; n = 10) and obese (35 ± 3 yrs; n = 11) subjects during either saline (control) or amino acid (AA) infusions. AA infusion increased ADP-stimulated respiration (i.e., coupled respiration), non-ADP stimulated respiration (i.e., uncoupled respiration), and ATP production rates in SS, but not IMF mitochondria in lean (n = 10; P < 0.05). Neither infusion increased any of the above parameters in muscle SS or IMF mitochondria of the obese subjects.
Using label free quantitative mass spectrometry, we determined differences in proteomes of SM SS and IMF mitochondria between lean (33 ± 3 yrs; n = 16) and obese (32 ± 3 yrs; n = 17) subjects. Differentially-expressed mitochondrial proteins in SS versus IMF mitochondria of obese subjects were associated with biological processes that regulate: electron transport chain (P<0.0001), citric acid cycle (P<0.0001), oxidative phosphorylation (P<0.001), branched-chain amino acid degradation, (P<0.0001), and fatty acid degradation (P<0.001). Overall, these findings show that obesity is associated with redistribution of key biological processes within the mitochondrial reticulum responsible for regulating energy metabolism in human skeletal muscle.
Polarography was used to determine functional differences in isolated SS and IMF mitochondria between lean (37 ± 3 yrs; n = 10) and obese (35 ± 3 yrs; n = 11) subjects during either saline (control) or amino acid (AA) infusions. AA infusion increased ADP-stimulated respiration (i.e., coupled respiration), non-ADP stimulated respiration (i.e., uncoupled respiration), and ATP production rates in SS, but not IMF mitochondria in lean (n = 10; P < 0.05). Neither infusion increased any of the above parameters in muscle SS or IMF mitochondria of the obese subjects.
Using label free quantitative mass spectrometry, we determined differences in proteomes of SM SS and IMF mitochondria between lean (33 ± 3 yrs; n = 16) and obese (32 ± 3 yrs; n = 17) subjects. Differentially-expressed mitochondrial proteins in SS versus IMF mitochondria of obese subjects were associated with biological processes that regulate: electron transport chain (P<0.0001), citric acid cycle (P<0.0001), oxidative phosphorylation (P<0.001), branched-chain amino acid degradation, (P<0.0001), and fatty acid degradation (P<0.001). Overall, these findings show that obesity is associated with redistribution of key biological processes within the mitochondrial reticulum responsible for regulating energy metabolism in human skeletal muscle.
ContributorsKras, Katon Anthony (Author) / Katsanos, Christos (Thesis advisor) / Chandler, Douglas (Committee member) / Dinu, Valentin (Committee member) / Mor, Tsafrir S. (Committee member) / Arizona State University (Publisher)
Created2017
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
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
Over 40% of adults in the United States are considered obese. Obesity is known to cause abnormal metabolic effects and lead to other negative health consequences. Interestingly, differences in metabolism and contractile performance between obese and healthy weight individuals are associated with differences in skeletal muscle fiber type composition between these groups. Each fiber type is characterized by unique metabolic and contractile properties, which are largely determined by the myosin heavy chain isoform (MHC) or isoform combination that the fiber expresses. In previous studies, SDS-PAGE single fiber analysis has been utilized as a method to determine MHC isoform distribution and single fiber type distribution in skeletal muscle. Herein, a methodological approach to analyze MHC isoform and fiber type distribution in skeletal muscle was fine-tuned for use in human and rodent studies. In the future, this revised methodology will be implemented to evaluate the effects of obesity and exercise on the phenotypic fiber type composition of skeletal muscle.
ContributorsOhr, Jalonna Rose (Author) / Katsanos, Christos (Thesis director) / Tucker, Derek (Committee member) / Serrano, Nathan (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05