Matching Items (3)
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- All Subjects: Metabolism
- All Subjects: Bioelectrochemistry
- 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
Exoelectrogenic microorganisms can grow by transferring electrons from their internal metabolism to extracellular substrates in a process known as extracellular electron transfer (EET). This dissertation explores the mechanisms of EET by both chemotrophic and phototrophic organisms and constructs a novel supramolecular structure that can be used as a model for microbial, long-range electron transfer. Geobacter sulfurreducens has been hypothesized to secrete and use riboflavin as a soluble, extracellular redox shuttle in conjunction with multi-heme, outer membrane, c-type cytochromes, but the required proteins and their properties have not been defined. To address the mechanism of extracellular electron transfer by G. sulfurreducens, the first part of this work explores the interaction between an outer membrane, octaheme, c-type cytochrome OmcZs from G. sulfurreducens and riboflavin. Interrogation via multiple physical techniques shows that OmcZs transfers electrons to riboflavin. By analogy to other characterized systems, riboflavin then likely interacts with extracellular acceptors directly. The second part of this work addresses the mechanisms of EET by the model cyanobacterium Synechocystis sp. PCC 6803. It has been hypothesized that Synechocystis employs conductive pili for production of extracellular current. However, the results herein show that a strain that does not have pili produces extracellular photocurrent in a direct electrochemical cell at a level similar to that by wild type cells. Furthermore, conductive atomic force microscopy (AFM) imaging is used to show that pili produced by the wild type organism are not conductive. Thus, an alternative EET mechanism must be operable. In the third part of this work, a supramolecular structure comprised of peptide and cytochromes designed to serve as a model for long-range electron transfer through cytochrome rich environments is described. The c-type cytochromes in this synthetic nanowire retain their redox activity after assembly and have suitable characteristics for long-range electron transfer. Taken together, the results of this dissertation not only inform on natural microbial mechanisms for EET but also provide a starting point to develop novel, synthetic systems.
ContributorsThirumurthy, Miyuki (Author) / Jones, Anne K (Thesis advisor) / Redding, Kevin (Committee member) / Torres, Cesar (Committee member) / Arizona State University (Publisher)
Created2019