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- All Subjects: Metabolism
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 removal of support material from metal 3D printed objects is a laborious necessity for the post-processing of powder bed fusion printing (PBF). Supports are typically mechanically removed by machining techniques. Sacrificial supports are necessary in PBF printing to relieve thermal stresses and support overhanging parts often resulting in the inclusion of supports in regions of the part that are not easily accessed by mechanical removal methods. Recent innovations in PBF support removal include dissolvable metal supports through an electrochemical etching process. Dissolvable PBF supports have the potential to significantly reduce the costs and time associated with traditional support removal. However, the speed and effectiveness of this approach is inhibited by numerous factors such as support geometry and metal powder entrapment within supports. To fully realize this innovative approach, it is necessary to model and understand the design parameters necessary to optimize support structures applicable to an electrochemical etching process. The objective of this study was to evaluate the impact of block additive manufacturing support parameters on key process outcomes of the dissolution of 316 stainless steel support structures. The parameters investigated included hatch spacing and perforation, and the outcomes of interests included time required for completion, surface roughness, and effectiveness of the etching process. Electrical current was also evaluated as an indicator of process completion. Analysis of the electrical current throughout the etching process showed that the dissolution is diffusion limited to varying degrees, and is dependent on support structure parameters. Activation and passivation behavior was observed during current leveling, and appeared to be more pronounced in non-perforated samples with less dense hatch spacing. The correlation between electrical current and completion of the etching process was unclear, as the support structures became mechanically removable well before the current leveled. The etching process was shown to improve surface finish on unsupported surfaces, but support was shown to negatively impact surface finish. Tighter hatch spacing was shown to correlate to larger variation in surface finish, due to ridges left behind by the support structures. In future studies, it is recommended current be more closely correlated to process completion and more roughness data be collected to identify a trend between hatch spacing and surface roughness.
ContributorsAbranovic, Brandon (Author) / Hildreth, Owen (Thesis director) / Torres, Cesar (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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