Matching Items (3)
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
Four enzymes, ATF1, ATF2, ATF, and CAT, were screened to determine which would be most effective at catalyzing the formation of aromatic esters. The CAT enzyme successfully catalyzed the reaction to produce 2-phenethyl acetate using 20x more lysate to improve the probability of enzyme presence in the lysate. The CAT enzyme was able to catalyze the reaction producing concentrations that increased by 62% every 12 hours. Enzymatic activity resulted in the production of 2.15 mg/L of 2-phenethyl acetate at 12 hours, 5.62 mg/L of 2-phenethyl acetate at 24 hours, and 15.12 mg/L of 2-phenethyl acetate at 48 hours.
ContributorsBrown, Kristen Ashley (Author) / Nielsen, David (Thesis director) / Thompson, Brian (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Aromatic compounds have traditionally been generated via petroleum feedstocks and have wide ranging applications in a variety of fields such as cosmetics, food, plastics, and pharmaceuticals. Substantial improvements have been made to sustainably produce many aromatic chemicals from renewable sources utilizing microbes as bio-factories. By assembling and optimizing native and non-native pathways to produce natural and non-natural bioproducts, the diversity of biochemical aromatics which can be produced is constantly being improved upon. One such compound, 2-Phenylethanol (2PE), is a key molecule used in the fragrance and food industries, as well as a potential biofuel. Here, a novel, non-natural pathway was engineered in Escherichia coli and subsequently evaluated. Following strain and bioprocess optimization, accumulation of inhibitory acetate byproduct was reduced and 2PE titers approached 2 g/L – a ~2-fold increase over previously implemented pathways in E. coli. Furthermore, a recently developed mechanism to
allow E. coli to consume xylose and glucose, two ubiquitous and industrially relevant microbial feedstocks, simultaneously was implemented and systematically evaluated for its effects on L-phenylalanine (Phe; a precursor to many microbially-derived aromatics such as 2PE) production. Ultimately, by incorporating this mutation into a Phe overproducing strain of E. coli, improvements in overall Phe titers, yields and sugar consumption in glucose-xylose mixed feeds could be obtained. While upstream efforts to improve precursor availability are necessary to ultimately reach economically-viable production, the effect of end-product toxicity on production metrics for many aromatics is severe. By utilizing a transcriptional profiling technique (i.e., RNA sequencing), key insights into the mechanisms behind styrene-induced toxicity in E. coli and the cellular response systems that are activated to maintain cell viability were obtained. By investigating variances in the transcriptional response between styrene-producing cells and cells where styrene was added exogenously, better understanding on how mechanisms such as the phage shock, heat-shock and membrane-altering responses react in different scenarios. Ultimately, these efforts to diversify the collection of microbially-produced aromatics, improve intracellular precursor pools and further the understanding of cellular response to toxic aromatic compounds, give insight into methods for improved future metabolic engineering endeavors.
allow E. coli to consume xylose and glucose, two ubiquitous and industrially relevant microbial feedstocks, simultaneously was implemented and systematically evaluated for its effects on L-phenylalanine (Phe; a precursor to many microbially-derived aromatics such as 2PE) production. Ultimately, by incorporating this mutation into a Phe overproducing strain of E. coli, improvements in overall Phe titers, yields and sugar consumption in glucose-xylose mixed feeds could be obtained. While upstream efforts to improve precursor availability are necessary to ultimately reach economically-viable production, the effect of end-product toxicity on production metrics for many aromatics is severe. By utilizing a transcriptional profiling technique (i.e., RNA sequencing), key insights into the mechanisms behind styrene-induced toxicity in E. coli and the cellular response systems that are activated to maintain cell viability were obtained. By investigating variances in the transcriptional response between styrene-producing cells and cells where styrene was added exogenously, better understanding on how mechanisms such as the phage shock, heat-shock and membrane-altering responses react in different scenarios. Ultimately, these efforts to diversify the collection of microbially-produced aromatics, improve intracellular precursor pools and further the understanding of cellular response to toxic aromatic compounds, give insight into methods for improved future metabolic engineering endeavors.
ContributorsMachas, Michael (Author) / Nielsen, David R (Thesis advisor) / Haynes, Karmella (Committee member) / Wang, Xuan (Committee member) / Nannenga, Brent (Committee member) / Varman, Arul (Committee member) / Arizona State University (Publisher)
Created2019