Matching Items (10)
- All Subjects: Metabolic Engineering
- Creators: Nielsen, David
- Creators: Torres, Cesar
- Member of: Theses and Dissertations
- Resource Type: Text
- Status: Published
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.
In our modern world the source of for many chemicals is to acquire and refine oil. This process is becoming an expensive to the environment and to human health. Alternative processes for acquiring the final product have been developed but still need work. One product that is valuable is butanol. The normal process for butanol production is very intensive but there is a method to produce butanol from bacteria. This process is better because it is more environmentally safe than using oil. One problem however is that when the bacteria produce too much butanol it reaches the toxicity limit and stops the production of butanol. In order to keep butanol from reaching the toxicity limit an adsorbent is used to remove the butanol without harming the bacteria. The adsorbent is a mesoporous carbon powder that allows the butanol to be adsorbed on it. This thesis explores different designs for a magnetic separation process to extract the carbon powder from the culture.
Flavonoids are important biomolecules with a variety of pharmaceutical and agricultural applications. Currently, isolating these compounds is done by plant extraction, however this process is hindered by large land and energy requirements. Previous groups have aimed to overcome these challenges by engineering microbes to produce these important compounds, however this is largely bottlenecked by the lack of intercellular malonyl-CoA availability. To remedy this, the genes matB and matC have been identified as coding for malonyl-CoA synthase and a putative dicarboxylate carrier protein, respectively. Other works have successfully engineered two variants, Streptomyces coelicolor and Rhizobium trifolii, of these genes into Escherichia coli, however this has yet to be accomplished in Gram-positive Corynebacterium glutamicum. Additionally, other groups have neglected to attempt tuning these genes with respect to one another by inserting in front of different inducible promoters. This study has successfully assembled two plasmids containing the Streptomyces coelicolor and Rhizobium trifolii variants of both matB and matC. Preliminary fermentations and GCMS results confirmed that little to none naringenin was produced without the matB-matC module. Additionally, preliminary fermentations revealed that the DelAro1 and DelAro3 strains can be used to reduce metabolism of aromatics like naringenin.
Escherichia coli is a bacterium that is used widely in metabolic engineering due to its ability to grow at a fast rate and to be cultured easily. E. coli can be engineered to produce many valuable chemicals, including biofuels and L-Phenylalanine—a precursor to many pharmaceuticals. Significant cell growth occurs in parallel to the biosynthesis of the desired biofuel or biochemical product, and limits product concentrations and yields. Stopping cell growth can improve chemical production since more resources will go toward chemical production than toward biomass. The goal of the project is to test different methods of controlling microbial uptake of nutrients, specifically phosphate, to dynamically limit cell growth and improve biochemical production of E. coli, and the research has the potential to promote public health, sustainability, and environment. This can be achieved by targeting phosphate transporter genes using CRISPRi and CRISPR, and they will limit the uptake of phosphate by targeting the phosphate transporter genes in DNA, which will stop transcriptions of the genes. In the experiment, NST74∆crr∆pykAF, a L-Phe overproducer, was used as the base strain, and the pitA phosphate transporter gene was targeted in the CRISPRi and CRISPR systems with the strain with other phosphate transporters knocked out. The tested CRISPRi and CRISPR mechanisms did not stop cell growth or improved L-Phe production. Further research will be conducted to determine the problem of the system. In addition, the CRISPRi and CRISPR systems that target multiple phosphate transporter genes will be tested in the future as well as the other method of stopping transcriptions of the phosphate transporter genes, which is called a tunable toggle switch mechanism.
Cyanobacteria have the potential to efficiently produce L-serine, an industrially important amino acid, directly from CO2 and sunlight, which is a more sustainable and inexpensive source of energy as compared to current methods. The research aims to engineer a strain of Cyanobacterium Synechococcus sp. PCC 7002 that increases L-serine production by mutating regulatory mechanisms that natively inhibit its production and encoding an exporter. While an excess of L-serine was not found in the supernatant of the cell cultures, with further fine tuning of the metabolic pathway and culture conditions, high titers of L-serine can be found. With the base strain engineered, the work can be extended and optimized by deleting degradation pathways, tuning gene expression levels, optimizing growth conditions, and investigating the effects of nitrogen supplementation for the strain.
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.
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.
One of the primary bottlenecks to chemical production in biological organisms is the toxicity of the chemical. Overexpression of efflux pumps has been shown to increase tolerance to aromatic compounds such as styrene and styrene oxide. Tight control of pump expression is necessary to maximize titers and prevent excessive strain on the cells. This study aimed to identify aromatic-sensitive native promoters and heterologous biosensors for construction of closed-loop control of efflux pump expression in E. coli. Using a promoter library constructed by Zaslaver et al., activation was measured through GFP output. Promoters were evaluated for their sensitivity to the addition of one of four aromatic compounds, their "leaking" of signal, and their induction threshold. Out of 43 targeted promoters, 4 promoters (cmr, mdtG, yahN, yajR) for styrene oxide, 2 promoters (mdtG, yahN) for styrene, 0 promoters for 2-phenylethanol, and 1 promoter for phenol (pheP) were identified as ideal control elements in aromatic bioproduction. In addition, a series of three biosensors (NahR, XylS, DmpR) known to be inducible by other aromatics were screened against styrene oxide, 2-phenylethanol, and phenol. The targeted application of these biosensors is aromatic-induced activation of linked efflux pumps. All three biosensors responded strongly in the presence of styrene oxide and 2-phenylethanol, with minor activation in the presence of phenol. Bioproduction of aromatics continues to gain traction in the biotechnology industry, and the continued discovery of aromatic-inducible elements will be essential to effective pathway control.
The field of bioprocess engineering has become an increasingly popular route to produce chemicals and fuels in a sustainable fashion. Bioprocessing is an interdisciplinary field that joins chemical engineering, metabolic engineering, and synthetic biology to tackle problems that will arise from the ongoing use of products derived from non-renewable resources. This study will overlook two effective tools that are widely used in the bioprocessing field. The first tool that was studied was strain optimization for biochemical production. This involves genetic manipulation of microbial hosts to create strains that are more efficient at producing the desired products. The second tool that was studied was adaptive laboratory evolution which is used to enhance overall cellular fitness. Enhancing the overall fitness and efficiency of these microbial production factories, allows for innovation and growth in the biochemical industry. Creating sustainable and renewable solutions for both specialty and commodity chemicals.
Strain optimization was specifically studied by enhancing inorganic carbon uptake in synechococcus sp. 7002. It is desired to have both high flux and high affinity transport for the rapid and efficient uptake of HCO3- for enhanced cell growth. The results found that the regulatory gene for carbon transporters in synechococcus genome was successfully deleted. Increasing the toxicity limits of 2-Phenylethanol was done by using adaptive laboratory evolution (ALE). ALE is a widely used practice in biotechnology studies to gain insights on mechanisms of molecular evolution and to better define the functionality of microbial cell factories. It was found that after growing E. coli BW25113 under selective conditions the genome evolved for a higher fitness medium with an increased concentration of 2-Phenylethanol. Overall, two key tools used in bioprocess engineering were successful studied to gain a better insight on the future of biochemical production industry.
Due to the wide range of health properties flavonoids possess, flavonoids are sold in health supplements to the general public. Flavonoids are also utilized in research but have a high cost due to current production techniques. This project focuses on engineering two DNA recombinants to develop new strains of Corynebacterium glutamicum that can produce flavonoids pinocembrin and naringenin. After culturing Escherichia coli colonies containing genes of interest, the genes were collected and purified by PCR reactions. The recombinant plasmid was assembled using CPEC and successfully transformed into Escherichia coli, with plans to transform Corynebacterium glutamicum to experiment and determine which recombinant can produce more pinocembrin and naringenin. Design work for other DNA recombinants, which were not the focus of this project, was also completed.