Matching Items (8)

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Literature Review to Analyze Pathway Bottlenecks for the Microbial Production of Flavonoids

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The development of Corynebacterium glutamicum for the microbial production of high-value products has made this bacterium an industrial workhorse. This metabolically engineered microbe is capable of accumulating and secreting flavonoids,

The development of Corynebacterium glutamicum for the microbial production of high-value products has made this bacterium an industrial workhorse. This metabolically engineered microbe is capable of accumulating and secreting flavonoids, a class of high functioning compounds found in plants. In human health, flavonoids are known to have powerful antioxidant, anti-inflammatory, anticancer, and antiviral properties which has led the growing interest to produce these compounds commercially. Recent literature seeks to overcome potential pathway bottlenecks to optimize flavonoid production by regulating protein expression within the central carbon, shikimate, chorismate, and fatty acid synthesis pathways. This paper reviews engineering strategies performed to increase the precursor titers of malonyl-CoA, phenylalanine, and tyrosine for increased flavonoid production.

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  • 2020-05

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Genetic Engineering of Cyanobacteria to Improve Photosynthetic Yield

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Increasing energy and environmental problems describe the need to develop renewable chemicals and fuels. Global research has been targeting using microbial systems on a commercial scale for synthesis of valuable

Increasing energy and environmental problems describe the need to develop renewable chemicals and fuels. Global research has been targeting using microbial systems on a commercial scale for synthesis of valuable compounds. The goal of this project was to refactor and overexpress b6-f complex proteins in cyanobacteria to improve photosynthesis under dynamic light conditions. Improvement in the photosynthetic system can directly relate to higher yields of valuable compounds such as carotenoids and higher yields of biomass which can be used as energy molecules. Four engineered strains of cyanobacteria were successfully constructed and overexpressed the corresponding four large subunits in the cytochrome b6-f complex. No significant changes were found in cell growth or pigment titer in the modified strains compared to the wild type. The growth assay will be performed at higher and/or dynamic light intensities including natural light conditions for further analysis.

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  • 2020-05

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Cheminformatic-based characterization of malate and lactate export networks

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Fermentative bioproduction is an efficient production avenue for many small organic acids with less greenhouse gas emissions than petrochemical conversion. Export of these organic acids from the cell is proposed

Fermentative bioproduction is an efficient production avenue for many small organic acids with less greenhouse gas emissions than petrochemical conversion. Export of these organic acids from the cell is proposed to be mediated by networks of transmembrane transport proteins. However characterization of full transporter networks or the substrate promiscuity of individual transporters is often incomplete. Here, we used a cheminformatic approach to predict previously unknown native activity of E. coli transporters based on substrate promiscuity. Experimental validation in characterized several major putative malate exporters, whereas others were characterized as weak putative lactate exporters. The lactate export network remains incompletely characterized and might be mediated by a large, evolved network of promiscuous transporters.

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Date Created
  • 2018-12

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Hydrothermal Liquefaction of Micro-Algae to Produce Liquid Biofuels

Description

Fossil fuels are currently the main source of energy in the world’s transportation sector. They are also the primary contributor to carbon emissions in the atmosphere, leading to adverse climate

Fossil fuels are currently the main source of energy in the world’s transportation sector. They are also the primary contributor to carbon emissions in the atmosphere, leading to adverse climate effects. The objective of the following research is to increase the yield and efficiency of algal biofuel in order to establish algal-derived fuel as a competitive alternative to predominantly used fossil fuels. Using biofuel commercially will reduce the cost of production and ultimately decrease additional carbon emissions. Experiments were performed using hydrothermal liquefaction (HTL) to determine which catalyst would enhance the algal biocrude oil and result in the highest quality biofuel product, as well as to find the optimal combination of processing temperature and manure co-liquefaction of biomass ratio. For the catalytic upgrading experiments, Micractenium Immerum algae was used in conjunction with pure H2, Pt/C, MO2C, and HZSM-5 catalysts at 350℃ and 400℃, 430 psi, and a 30-minute residence time to investigate the effects of catalyst choice and temperature on the crude oil yield. While all catalysts increased the carbon content of the crude oil, it was found that using HZSM-5 at 350℃ resulted in the greatest overall yield of about 75%. However, the Pt/C catalyst increased the HHV from 34.26 MJ/kg to 43.26 MJ/kg. Cyanidioschyzon merolae (CM) algae and swine manure were utilized in the co-liquefaction experiments, in ratios (algae to swine) of 80:20, 50:50, and 20:80 at temperatures of 300℃ and 330℃. It was found that a ratio of 80:20 at 330℃ produced the highest biocrude oil yield of 29.3%. Although the 80:20 experiments had the greatest biomass conversion and best supported the deacidification of the oil product, the biocrude oil had a HHV of 33.58 MJ/kg, the lowest between the three different ratios. However, all calorific values were relatively close to each other, suggesting that both catalytic upgrading and co-liquefaction can increase the efficiency and economic viability of algal biofuel.

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  • 2020-05

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Development of a Polyethylene terephthalate Hydrolase Expression System in Escherichia coli

Description

The increased shift towards environmentalism has brought notable attention to a universal excessive plastic consumption and subsequent plastic overload in landfills. Among these plastics, polyethylene terephthalate, more commonly known as

The increased shift towards environmentalism has brought notable attention to a universal excessive plastic consumption and subsequent plastic overload in landfills. Among these plastics, polyethylene terephthalate, more commonly known as PET, constitutes a large percentage of the waste that ends up in landfills. Material and chemical/thermal methods for recycling are both costly, and inefficient, which necessitates a more sustainable and cheaper alternative. The current study aims at fulfilling that role through genetic engineering of Bacillus subtilis with integration of genes from LCC, Ideonella sakaiensis, and Bacillus subtilis. The plasmid construction was done through restriction cloning. A recombinant plasmid for the expression of LCC was constructed, and transformed into Escherichia coli. Future experiments for this study should include redesigning of primers, with possible combination of signal peptides with genes during construct design, and more advanced assays for effective outcomes.

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  • 2021-05

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Enhancing Escherichia coli Fermentative Performance with Lactobacillus plantarum WCFS1 Genes

Description

Renewable bioproduction through fermentation of microbial species such as E. coli shows much promise in comparison to conventional fossil fuel based chemical production. Although Escherichia coli is a workhorse for

Renewable bioproduction through fermentation of microbial species such as E. coli shows much promise in comparison to conventional fossil fuel based chemical production. Although Escherichia coli is a workhorse for bioproduction, there are inherent limitations associated with the use of this organism which negatively affect bioproduction. One example is E. coli fermentative growth being less robust compared to some microbes such as Lactobacilli under anaerobic and microaerobic fermentation conditions. Identification and characterization of its fermentative growth constraints will help in making E. coli a better fermentation host. In this thesis, I demonstrate that Lactobacillus plantarum WCFS1 has desirable fermentative capabilities that may be transferrable to E. coli through genetic engineering to alleviate growth restraints. This has led to the hypothesis that these L. plantarum DNA sequences are transferrable through a genomic library. A background of comparative genomics and complementary literature review has demonstrated that E. coli growth may be hindered by stress from many toxin-antitoxin systems. L. plantarum WCFS1 optimizes amino acid catabolism over glycolysis to generate high ATP levels from reducing agents and proton motive force, and Lactobacilli are resistant to acidic environments and encodes a wide variety of acid transporters that could help E. coli fermentative growth. Since a great variety of L. plantarum genes may contribute to its fermentative capabilities, a gDNA library containing L. plantarum WCFS1 genes has been successfully constructed for testing in E. coli bioproducers to search for specific genes that may enhance E. coli fermentative performance and elucidate the molecular basis of Lactobacillus fermentative success.

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Date Created
  • 2019-05

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Investigating strategies to enhance microbial production of and tolerance towards aromatic biochemicals

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

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.

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Date Created
  • 2019

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Expression, Characterization, and Structural Studies of Anti-amyloid Antibody Fragments

Description

Alzheimer’s disease is a major problem affecting over 5.7 million Americans. Although much is known about the effects of this neurogenerative disease, the exact pathogenesis is still unknown. One very

Alzheimer’s disease is a major problem affecting over 5.7 million Americans. Although much is known about the effects of this neurogenerative disease, the exact pathogenesis is still unknown. One very important characteristic of Alzheimer’s is the accumulation of beta amyloid protein which often results in plaques. To understand these beta amyloid proteins better, antibody fragments may be used to bind to these oligomers and potentially reduce the effects of Alzheimer’s disease.

This thesis focused on the expression and crystallization the fragment antigen binding antibody fragment A4. A fragment antigen binding fragment was chosen to be worked with as it is more stable than many other antibody fragments. A4 is important in Alzheimer’s disease as it is able to identify toxic beta amyloid.

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Date Created
  • 2018