Matching Items (34)
Description
The symbiosis between termites and their parabasalid hindgut protists centers around the wood digestion that is needed for both species to acquire the nutrients from wood. One of the important carbohydrate-active proteins required for the wood breakdown are glycoside hydrolase (GH) families. Previous studies have looked at the phylogeny of some of these protein families from a termite whole gut transcriptome or in a different context than lignocellulose digestion. In this study, we attempt to understand the function and evolution of these GH families in the context of protist evolution by using protist single cell transcriptomes. 14 families of interest were chosen to create phylogenetic trees: GH2, GH3, GH5, GH7, GH8, GH9, GH10, GH11, GH26, GH43, GH45, GH55, GH67, GH95 for their interesting expressions across different protists such as being present in all protists or being present in only termite-associated protists. The dbCAN2 (automated Carbohydrate-active enzyme ANnotation) program was used to find GH families in each of the protist single cell transcriptomes and additional characterized sequences registered on the National Center for Biotechnology Information to create phylogenetic trees for each of the GH families of interest. Results show that many of the GH families expressed in protists were acquired through horizontal gene transfer from fungi and bacteria. Additionally, comparison to the parabasalid phylogeny indicates most GH families evolved independently from the protists. Based on the pattern of expression of these GH families throughout different protist orders, conclusions can be made about whether the specific family was vertically or horizontally acquired in the termite symbionts.
ContributorsJahan, Israa (Author) / Gile, Gillian (Thesis director) / Wang, Xuan (Committee member) / Swichtenberg, Kali (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Department of Psychology (Contributor)
Created2023-05
Description
Lignocellulose, the major structural component of plant biomass, represents arenewable substrate of enormous biotechnological value. Microbial production of
chemicals from lignocellulosic biomass is an attractive alternative to chemical synthesis.
However, to create industrially competitive strains to efficiently convert lignocellulose to
high-value chemicals, current challenges must be addressed. Redox constraints, allosteric
regulation, and transport-related limitations are important bottlenecks limiting the
commercial production of renewable chemicals from lignocellulose. Advances in
metabolic engineering techniques have enabled researchers to engineer microbial strains
that overcome some of these challenges but new approaches that facilitate the
commercial viability of lignocellulose valorization are needed. Biological systems are
complex with a plethora of regulatory systems that must be carefully modulated to
efficiently produce and excrete the desired metabolites. In this work, I explore metabolic
engineering strategies to address some of the biological constraints limiting
bioproduction such as redox, allosteric, and transport constraints to facilitate cost-effective
lignocellulose bioconversion.
ContributorsOnyeabor, Moses Ekenedilichukwu (Author) / Wang, Xuan (Thesis advisor) / Varman, Arul M (Committee member) / Nannenga, Brent (Committee member) / Nielsen, David R (Committee member) / Geiler-Samerotte, Kerry (Committee member) / Arizona State University (Publisher)
Created2023
Characterization and Manipulation of Microbiomes From Arid Landfills for Improved Methane Production
Description
Environmentally harmful byproducts from solid waste’s decomposition, including methane (CH4) emissions, are managed through standardized landfill engineering and gas-capture mechanisms. Yet only a limited number of studies have analyzed the development and composition of Bacteria and Archaea involved in CH4 production from landfills. The objectives of this research were to compare microbiomes and bioactivity from CH4-producing communities in contrasting spatial areas of arid landfills and to tests a new technology to biostimulate CH4 production (methanogenesis) from solid waste under dynamic environmental conditions controlled in the laboratory. My hypothesis was that the diversity and abundance of methanogenic Archaea in municipal solid waste (MSW), or its leachate, play an important role on CH4 production partially attributed to the group’s wide hydrogen (H2) consumption capabilities. I tested this hypothesis by conducting complementary field observations and laboratory experiments. I describe niches of methanogenic Archaea in MSW leachate across defined areas within a single landfill, while demonstrating functional H2-dependent activity. To alleviate limited H2 bioavailability encountered in-situ, I present biostimulant feasibility and proof-of-concepts studies through the amendment of zero valent metals (ZVMs). My results demonstrate that older-aged MSW was minimally biostimulated for greater CH4 production relative to a control when exposed to iron (Fe0) or manganese (Mn0), due to highly discernable traits of soluble carbon, nitrogen, and unidentified fluorophores found in water extracts between young and old aged, starting MSW. Acetate and inhibitory H2 partial pressures accumulated in microcosms containing old-aged MSW. In a final experiment, repeated amendments of ZVMs to MSW in a 600 day mesocosm experiment mediated significantly higher CH4 concentrations and yields during the first of three ZVM injections. Fe0 and Mn0 experimental treatments at mesocosm-scale also highlighted accelerated development of seemingly important, but elusive Archaea including Methanobacteriaceae, a methane-producing family that is found in diverse environments. Also, prokaryotic classes including Candidatus Bathyarchaeota, an uncultured group commonly found in carbon-rich ecosystems, and Clostridia; All three taxa I identified as highly predictive in the time-dependent progression of MSW decomposition. Altogether, my experiments demonstrate the importance of H2 bioavailability on CH4 production and the consistent development of Methanobacteriaceae in productive MSW microbiomes.
ContributorsReynolds, Mark Christian (Author) / Cadillo-Quiroz, Hinsby (Thesis advisor) / Krajmalnik-Brown, Rosa (Thesis advisor) / Wang, Xuan (Committee member) / Kavazanjian, Edward (Committee member) / Arizona State University (Publisher)
Created2022
Description
The current use of non-renewable fossil fuels for industry poses a threat for future generations. Thus, a pivot to renewable sources of energy must be made to secure a
sustainable future. One potential option is the utilization of metabolically engineered
bacteria to produce value-added chemicals during fermentation. Currently, numerous
strains of metabolically engineered Escherichia coli have shown great capacity to
specialize in the production of high titers of a desired chemical. These metabolic systems,
however, are constrained by the biological limits of E. coli itself. During fermentation, E.
coli grows to less than one twentieth of the density that aerobically growing cultures can
reach. I hypothesized that this decrease in growth during fermentation is due to cellular
stress associated with fermentative growth, likely caused by stress related genes. These
genes, including toxin-antitoxin (TA) systems and the rpoS mediated general stress
response, may have an impact on fermentative growth constraints. Through
transcriptional analysis, I identified that the genes pspC and relE are highly expressed in
fermenting strains of both wild type and metabolically engineered E. coli. Fermentation
of toxin gene knockouts of E. coli BW25113 revealed their potential impacts on E. coli
fermentation. The inactivation of ydcB, lar, relE, hipA, yjfE, chpA, ygiU, ygjN, ygfX,
yeeV, yjdO, yjgK and ydcX did not lead to significant changes in cell growth when tested
using sealed tubes under microaerobic conditions. In contrast, inactivation of pspC, yafQ,
yhaV, yfjG and yoeB increased cell growth after 12 hours while inactivation yncN
significantly arrested cell growth in both tube and fermentation tests, thus proving these
toxins’ roles in fermentative growth. Moreover, inactivation of rpoS also significantly hindered the ability of E. coli to ferment, suggesting its important role in E. coli
fermentation
ContributorsHernandez, Michaella (Author) / Wang, Xuan (Thesis advisor) / Nielsen, David (Committee member) / Varman, Arul (Committee member) / Arizona State University (Publisher)
Created2022
Description
Single and double deletion strains of Escherichia coli were grown in paired co-cultures with an intent to identify examples of metabolite exchange and cooperative interactions between strains. The essential genes pheA, argA, tyrA, and trpC, as well as the non- essential genes pykF, pykA, mdh, ppc, and nuoN were deleted from Escherichia coli strains Bw25113 and ATCC 9637. Cultures were paired at three different initial ratios and grown at plate and flask scale. Optical density measurements were used to observe the performance of tested co-cultures, with changes in maximum optical density and growth rate used as indicators of interaction or lack thereof between tested pairs. Auxotrophic strains unable to produce essential amino acids were observed to grow in co-culture but not in monoculture, indicative of metabolite exchange facilitating growth. An increase in optical density for non-essential pairs when compared to the prototrophic parent and precursor monocultures was indicative of metabolite exchange. The initial frequency of paired mutants with non-essential deletions appeared to have an impact on growth performance, but whether this was indicative of any beneficial exchange was not able to be determined from data.
ContributorsFenner, Alexander James (Author) / Nielsen, David (Thesis advisor) / Wang, Xuan (Committee member) / Varman, Arul (Committee member) / Arizona State University (Publisher)
Created2022
Description
Biomass synthesis is a competing factor in biological systems geared towards generation of commodity and specialty chemicals, ultimately limiting maximum titer and yield; in this thesis, a widely generalizable, modular approach focused on decoupling biomass synthesis from the production of the phenylalanine in a genetically modified strain of E. coli BW25113 was explored with the use of synthetic trans-encoded small RNA (sRNA) to achieve greater efficiency. The naturally occurring sRNA MicC was used as a scaffold, and combined on a plasmid with a promoter for anhydrous tetracycline (aTc) and a T1/TE terminator. The coding sequence corresponding to the target binding site for fourteen potentially growth-essential gene targets as well as non-essential lacZ was placed in the seed region of the of the sRNA scaffold and transformed into BW25113, effectively generating a unique strain for each gene target. The BW25113 strain corresponding to each gene target was screened in M9 minimal media; decreased optical density and elongated cell morphology changes were observed and quantified in all induced sRNA cases where growth-essential genes were targeted. Six of the strains targeting different aspects of cell division that effectively suppressed growth and resulted in increased cell size were then screened for viability and metabolic activity in a scaled-up shaker flask experiment; all six strains were shown to be viable during stationary phase, and a metabolite analysis showed increased specific glucose consumption rates in induced strains, with unaffected specific glucose consumption rates in uninduced strains. The growth suppression, morphology and metabolic activity of the induced strains in BW25113 was compared to the bacteriostatic additives chloramphenicol, tetracycline, and streptomycin. At this same scale, the sRNA plasmid targeting the gene murA was transformed into BW25113 pINT-GA, a phenylalanine overproducer with the feedback resistant genes aroG and pheA overexpressed. Two induction times were explored during exponential phase, and while the optimal induction time was found to increase titer and yield amongst the BW25113 pINT-GA murA sRNA variant, overall this did not have as great a titer or yield as the BW25113 pINT-GA strain without the sRNA plasmid; this may be a result of the cell filamentation.
ContributorsHerschel, Daniel Jordan (Author) / Nielsen, David R (Thesis advisor) / Torres, César I (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2016
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
Description
Metabolic engineering of bacteria has become a viable technique as a sustainable and efficient method for the production of biochemicals. Two main goals were explored: investigating styrene tolerance genes in E. coli and engineering cyanobacteria for the high yield production of L-serine. In the first study, genes that were shown to be highly differentially expressed in E. coli upon styrene exposure were further investigated by testing the effects of their deletion and overexpression on styrene tolerance and growth. It was found that plsX, a gene responsible for the phospholipid formation in membranes, had the most promising results when overexpressed at 10 µM IPTG, with a relative OD600 of 706 ± 117% at 175 mg/L styrene when compared to the control plasmid at the same concentration. This gene is likely to be effective target when engineering styrene- and other aromatic-producing strains, increasing titers by reducing their cytotoxicity.In the second study, the goal is to engineer the cyanobacterium Synechococcus sp. PCC 7002 for the overproduction of L-serine. As a robust, photosynthetic bacteria, it has potential for being used in such-rich states to capture CO2 and produce industrially relevant products. In order to increase L-serine titers, a key degradation gene, ilvA, must be removed. While ilvA is responsible for degrading L-serine into pyruvate, it is also responsible for initiating the only known pathway for the production of isoleucine. Herein, we constructed a plasmid containing the native A0730 gene in order to investigate its potential to restore isoleucine production. If functional, a Synechococcus sp. PCC 7002 ΔilvA strain can then be engineered with minimal effects on growth and an expected increase in L-serine accumulation.
ContributorsAbed, Omar (Author) / Nielsen, David R (Thesis advisor) / Varman, Arul M (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2021
Description
Bioconversion of lignocellulosic sugars is often suboptimal due to global regulatory mechanisms such as carbon catabolite repression and incomplete/inefficient metabolic pathways. While conventional bioprocessing strategies for metabolic engineering have predominantly focused on a single engineered strain, the alternative development of synthetic microbial communities facilitates the execution of complex metabolic tasks by exploiting unique community features (i.e., modularity, division of labor, and facile tunability). In this dissertation, these features are leveraged to develop a suite of generalizable strategies and transformative technologies for engineering Escherichia coli coculture systems to more efficiently utilize lignocellulosic sugar mixtures. This was achieved by rationally pairing and systematically engineering catabolically-orthogonal Escherichia coli sugar specialists. Coculture systems were systematically engineered, as derived from either wild-type Escherichia coli W, ethanologenic LY180, lactogenic TG114 or succinogenic KJ122. Net catabolic activities were then readily balanced by simple tuning of the inoculum ratio between sugar specialists, ultimately enabling improved co-utilization (98% of 100 g L-1 total sugars) of glucose-xylose mixtures (2:1 by mass) under simple batch fermentation conditions. We next extended this strategy to a coculture-coproduction system capable of capturing and fixing CO2 evolved during biofuel production through inter-strain metabolic cooperation. Holistically, this work contributes to an improved understanding of the dynamic behavior of synthetic microbial consortia as enhanced bioproduction platforms and carbon conservation strategy for renewable fuels and chemicals from non-food carbohydrates
ContributorsFlores, Andrew David (Author) / Nielsen, David R (Thesis advisor) / Wang, Xuan (Thesis advisor) / Varman, Arul M (Committee member) / Nannenga, Brent (Committee member) / Wheeldon, Ian (Committee member) / Arizona State University (Publisher)
Created2021
Description
Abiotic stresses, such as heat, can drive protein misfolding and aggregation, leading to inhibition of cellular function and ultimately cell death. Unexpectedly, a thermotolerant Escherichia coli was identified from a pool of antibiotic resistant RNA polymerase β subunit (rpoB) mutants. This stress tolerant phenotype was characterized through exposure to high temperature and ethanol. After 30-minute exposure of cells to 55°C or 25% ethanol, the mutant displayed 100 times greater viability than the wild-type, indicating that the rpoB mutation may have broadly affected the cellular environment to reduce protein misfolding and/or prevent protein aggregation. To further test this hypothesis, we examined thermotolerance of cells lacking heat shock chaperone DnaJ (Hsp40), which is a cochaperone of one of the most abundant and conserved chaperones, DnaK (Hsp70). The deletion of dnaJ led to severe growth defects in the wild-type, namely a slower growth rate and extreme filamentation at 42°C. The severity of the growth defects increased after additionally deleting DnaJ analog, CbpA. However, these defects were significantly ameliorated by the rpoB mutation. Finally, the rpoB mutant was found to be minimally affected by the simultaneous depletion of DnaK and DnaJ compared to the wild-type, which failed to form single colonies at 37°C and 42°C. Based on these observations, it is proposed that the rpoB mutant’s robust thermotolerant phenotype results from a cellular environment protective against protein aggregation or improper folding. The folding environment of the rpoB mutants should be further examined to elucidate the mechanism by which both antibiotic resistance and thermotolerance can be conferred.
ContributorsYeh, Melody (Author) / Misra, Rajeev (Thesis director) / Wang, Xuan (Committee member) / Kelly, Keilen (Committee member) / School of Life Sciences (Contributor) / School of International Letters and Cultures (Contributor) / School of Human Evolution & Social Change (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05