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Description
p-Coumaric acid is used in the food, pharmaceutical, and cosmetic industries due to its versatile properties. While prevalent in nature, harvesting the compound from natural sources is inefficient, requiring large quantities of producing crops and numerous extraction and purification steps. Thus, the large-scale production of the compound is both difficult

p-Coumaric acid is used in the food, pharmaceutical, and cosmetic industries due to its versatile properties. While prevalent in nature, harvesting the compound from natural sources is inefficient, requiring large quantities of producing crops and numerous extraction and purification steps. Thus, the large-scale production of the compound is both difficult and costly. This research aims to produce p-coumarate directly from renewable and sustainable glucose using a co-culture of Yeast and E. Coli. Methods used in this study include: designing optimal media for mixed-species microbial growth, genetically engineering both strains to build the production pathway with maximum yield, and analyzing the presence of p-Coumarate and its pathway intermediates using High Performance Liquid Chromatography (HPLC). To date, the results of this project include successful integration of C4H activity into the yeast strain BY4741 ∆FDC1, yielding a strain that completely consumed trans-cinnamate (initial concentration of 50 mg/L) and produced ~56 mg/L p-coumarate, a resting cell assay of the co-culture that produced 0.23 mM p-coumarate from an initial L-Phenylalanine concentration of 1.14 mM, and toxicity tests that confirmed the toxicity of trans-cinnamate to yeast for concentrations above ~50 mg/L. The hope for this project is to create a feasible method for producing p-Coumarate sustainably.
ContributorsJohnson, Kaleigh Lynnae (Author) / Nielsen, David (Thesis director) / Thompson, Brian (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
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Description
The engineering of microbial cell factories capable of synthesizing industrially relevant chemical building blocks is an attractive alternative to conventional petrochemical-based production methods. This work focuses on the novel and enhanced biosynthesis of phenol, catechol, and muconic acid (MA). Although the complete biosynthesis from glucose has been previously demonstrated for

The engineering of microbial cell factories capable of synthesizing industrially relevant chemical building blocks is an attractive alternative to conventional petrochemical-based production methods. This work focuses on the novel and enhanced biosynthesis of phenol, catechol, and muconic acid (MA). Although the complete biosynthesis from glucose has been previously demonstrated for all three compounds, established production routes suffer from notable inherent limitations. Here, multiple pathways to the same three products were engineered, each incorporating unique enzyme chemistries and/or stemming from different endogenous precursors. In the case of phenol, two novel pathways were constructed and comparatively evaluated, with titers reaching as high as 377 ± 14 mg/L at a glucose yield of 35.7 ± 0.8 mg/g. In the case of catechol, three novel pathways were engineered with titers reaching 100 ± 2 mg/L. Finally, in the case of MA, four novel pathways were engineered with maximal titers reaching 819 ± 44 mg/L at a glucose yield of 40.9 ± 2.2 mg/g. Furthermore, the unique flexibility with respect to engineering multiple pathways to the same product arises in part because these compounds are common intermediates in aromatic degradation pathways. Expanding on the novel pathway engineering efforts, a synthetic ‘metabolic funnel’ was subsequently constructed for phenol and MA, wherein multiple pathways were expressed in parallel to maximize carbon flux toward the final product. Using this novel ‘funneling’ strategy, maximal phenol and MA titers exceeding 0.5 and 3 g/L, respectively, were achieved, representing the highest achievable production metrics products reported to date.
ContributorsThompson, Brian (Author) / Nielsen, David R (Thesis advisor) / Nannenga, Brent (Committee member) / Green, Matthew (Committee member) / Wang, Xuan (Committee member) / Moon, Tae Seok (Committee member) / Arizona State University (Publisher)
Created2017
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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

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
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Description

The R-specific alcohol dehydrogenase (ADH) from Lactobacillus brevis LB19 (LbADH) was studied with respect to its ability to reduce a series of 3- through 5-carbon 2-alkanones and aldehydes of relevance as biofuel precursors. Although active on all substrates tested, LbADH displays a marked preference for longer chain substrates. Interestingly, however,

The R-specific alcohol dehydrogenase (ADH) from Lactobacillus brevis LB19 (LbADH) was studied with respect to its ability to reduce a series of 3- through 5-carbon 2-alkanones and aldehydes of relevance as biofuel precursors. Although active on all substrates tested, LbADH displays a marked preference for longer chain substrates. Interestingly, however, 2-alkanones were found to impose substrate inhibition towards LbADH, whereas aldehyde substrates rendered no such effect. Inhibition caused by 2-alkanones was furthermore found to intensify with increasing chain length. Despite demonstrating both primary and secondary ADH activities, a preliminary sequence analysis suggests that LbADH remains distinct from other, previously characterized primary-secondary ADHs. In addition to further characterizing the substrate range of this industrially important enzyme, this study suggests that LbADH has the potential to serve as a useful enzyme for the engineering of various novel alcohol biofuel pathways.

ContributorsHalloum, Ibrahim (Author) / Thompson, Brian (Author) / Pugh, Shawn (Author) / Nielsen, David (Author) / Ira A. Fulton Schools of Engineering (Contributor)
Created2015-08-05