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Dissertations / Theses on the topic 'Rational Strain, Metabolic Engineering'

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Published: 6 September 2023

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1

Carere, Robert Carlo. "Genomics of cellulolytic clostridia and development of rational metabolic engineering strategies." MPI Open Access Journals, 2008. http://hdl.handle.net/1993/21707.

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Consolidated bioprocessing, a process in which cellulase production, substrate hydrolysis, and fermentation occur simultaneously, offers the potential for lower biofuel production costs than traditional approaches and is an economically attractive near-term goal for fermentative production of ethanol and/or hydrogen (H2) as biofuels. Current yields fall short of theoretical maxima, vary considerably between species, and are influenced by the highly branched metabolic pathways utilized by fermentative organisms. For fermentative ethanol/ H2 production to become practical, yields must be increased either through intelligent species selection, a manipulation of culture conditions, or via the implementation of rational metabolic engineering strategies. A comparative genomics approach amoungst select members of the Firmicutes, Euryarchaeota, and Thermotogae was used to identify genes relevent to ethanol and H2 production. Growth, end-product synthesis, enzyme activities and the associated transcription of select genes were studied in the cellulolytic anaerobe, Clostridium thermocellum ATCC 27405, during batch fermentation of cellobiose to determine the effect of elevated N2 and H2 sparging on end-product distribution. The absence of genes encoding acetaldehyde dehydrogenase and bifunctional acetaldehyde/alcohol dehydrogenase (AdhE) correlates with elevated H2 yields and low ethanol production. The type(s) of encoded hydrogenases appear to have minimal impact on H2 production in organisms that do not encode ethanologenic pathways, however, they do influence reduced end-product yields in those that do. We also find that while gas sparging can be used to effectively shift carbon and electron flow, the observed shifts at the pyruvate branch-point are likely principally influenced by the availability of reduced electron carriers (NAD, NADP, ferredoxin) and thermodynamic considerations. Finally, both electrotransformation and conjugative plasmid protocols were developed and evaluated for thermophilic species C. thermocellum and Thermoanaerobacter pseudethanolicus 39E, and the mesophilic bacterium, Clostridium termitidis CT1112. The efficiency of transformation for C. thermocellum strain ATCC 27405 is consistently low whereas transformation frequencies were ~100-fold higher in C. termitidis. Observed frequencies of plasmid transfer, via conjugation, were similar in both C. thermocellum and C. termitidis suggesting the transfer of single stranded DNA may circumvent aggressive restriction methylation systems.
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2

Chen, Lin [Verfasser]. "Rational Metabolic Engineering and Systematic Analysis of Escherichia coli for L-Tryptophan Bioproduction / Lin Chen." München : Verlag Dr. Hut, 2017. http://d-nb.info/1128466961/34.

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3

Schiefelbein, Sarah [Verfasser], and Christoph [Akademischer Betreuer] Wittmann. "Improved L-lysine production in Corynebacterium glutamicum by rational strain engineering / Sarah Schiefelbein ; Betreuer: Christoph Wittmann." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2015. http://d-nb.info/112757969X/34.

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4

Hills, Christopher. "Acetate metabolism in Geobacillus thermoglucosidasius and strain engineering for enhanced bioethanol production." Thesis, University of Bath, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.665397.

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Social, economic and political pressures have driven the development of renewable alternatives to fossil fuels. Biofuels, such as bioethanol, have proved to be successful alternatives. Mature technologies are crop-based, but this has brought criticism due to the conflicting use of land for fuel versus food production. Therefore, bioethanol production technologies have shifted to utilising the sugars that derive from the degradation of lignocellulosic biomass. The thermophilic, Gram-positive bacterium, Geobacillus thermoglucosidasius, can naturally utilise a large fraction of these sugars, and metabolic engineering has been used to create a strain that produces ethanol as the major product of fermentation. This strain, G. thermoglucosidasius TM242 (Δldh, Δpfl, pdhup), does however, produce small but significant quantities of acetate, an undesirable by-product of fermentation. Therefore, acetate metabolism in the G. thermoglucosidasius TM242 strain was the focus of this study. During fermentation, ethanol is generated from the central metabolite acetyl-CoA through the activities of a bifunctional enzyme: aldehyde dehydrogenase/alcohol dehydrogenase (ADHE). On the other hand, acetate is generated from acetyl-CoA through catalysis by phosphotransacetylase (PTA) and acetate kinase (AK). Acetate metabolism in G. thermoglucosidasius TM242 was studied in this project by investigating the enzyme activities governing flux from acetyl-CoA, and the feasibility of reduced acetate production was investigated by a pta-deletion strategy. This thesis reports the characterisation of PTA and AK, by studying activities from both native cell lysates and recombinantly expressed proteins. The results indicate that the activities of PTA and AK are greater than those of ADHE, suggesting that the potential metabolic flux is greater towards acetate production than to ethanol. However, the ethanol yield from G. thermoglucosidasius TM242 fermentations is greater than that of acetate, suggesting the existence of a regulatory mechanism controlling acetyl-CoA flux. Several possible regulatory mechanisms were studied in this project and are reported here. The viability of creating a strain that reduces acetate accumulation, and potentially increases ethanol yields, was investigated and reported in this thesis. The gene encoding PTA was deleted from G. thermoglucosidasius TM242, and the resulting strain was characterised. The Δpta strain had approximately 5% of the PTA activity measured in TM242, but acetate was still generated from pentose and hexose fermentations. Additional phosphotransacylase (PTAC) enzymes were discovered in G. thermoglucosidasius TM242 that could catalyse the conversion of acetyl-CoA and orthophosphate to acetyl-phosphate and CoA. A series of PTAC null strains were created and analysed, the results of which indicated that phosphotransbutyrylase (PTB) could be involved in acetate production in vivo. It was discovered that the cell lysates of G. thermoglucosidasius strains carrying deletions to both pta and ptb could no longer catalyse the conversion of acetyl-CoA and orthophosphate to acetyl-phosphate and CoA. However, these strains still accumulated acetate, suggesting the presence of alternative acetate-producing pathways in this organism. In addition, G. thermoglucosidasius strains carrying deletions to both pta and ptb could ferment glucose but not xylose, suggesting that the production of ATP by the PTA-AK pathway is crucial for micro-aerobic growth on pentose sugars.
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5

Gerebring, Linnéa. "Yeast Saccharomyces cerevisiae strain isolated from lager beer shows tolerance to isobutanol." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-129066.

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The development of biofuels has received much attention due to the global warming and limited resources associated with fossil fuels. Butanol has been identified as a potential option due to its advantages over ethanol, for example higher energy density, compatibility with current infrastructure and its possibility to be blended with gasoline at any ratio. Yeast Saccharomyces cerevisiae can be used as a producer of butanol. However, butanol toxicity to the host limits the yield produced. In this study, four strains of yeast isolated from the habitats of lager beer, ale, wine and baker ́s yeast were grown in YPD media containing isobutanol concentrations of 1.5 %, 2 %, 3 % and 4 %. Growth was measured to determine the most tolerant strain. Gene expression for the genes RPN4, RTG1 and ILV2 was also measured, to determine its involvement in butanol stress. The genes have in previous studies seen to be involved in butanol tolerance or production, and the hypothesis was that they all should be upregulated in response to butanol exposure. It was found that the yeast strain isolated from lager beer was most tolerant to isobutanol concentrations of 2 % and 3 %. It was also found that the gene RPN4 was upregulated in response to isobutanol stress. There was no upregulation of RTG1 or ILV2, which was unexpected. The yeast strain isolated from lager beer and the gene RPN4 is proposed to be investigated further, to be able to engineer a suitable producer of the biofuel butanol.
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6

Bi, Changhao. "Metabolic characterization and engineering of Enterobacter asburiae strain JDR-1 to develop microbial biocatalysts for efficient hemicellulose utilization." [Gainesville, Fla.] : University of Florida, 2009. http://purl.fcla.edu/fcla/etd/UFE0024266.

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7

Kachel, Benjamin [Verfasser], and Michael [Akademischer Betreuer] Lanzer. "Metabolic engineering of Synechococcus sp. strain PCC 7002 for the photoautotrophic production of riboflavin (vitamin B2) / Benjamin Kachel ; Betreuer: Michael Lanzer." Heidelberg : Universitätsbibliothek Heidelberg, 2021. http://nbn-resolving.de/urn:nbn:de:bsz:16-heidok-305364.

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8

Freedman, Benjamin Gordon. "Degenerate oligonucleotide primed amplification of genomic DNA for combinatorial screening libraries and strain enrichment." Diss., Virginia Tech, 2014. http://hdl.handle.net/10919/71346.

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Combinatorial approaches in metabolic engineering can make use of randomized mutations and/or overexpression of randomized DNA fragments. When DNA fragments are obtained from a common genome or metagenome and packaged into the same expression vector, this is referred to as a DNA library. Generating quality DNA libraries that incorporate broad genetic diversity is challenging, despite the availability of published protocols. In response, a novel, efficient, and reproducible technique for creating DNA libraries was created in this research based on whole genome amplification using degenerate oligonucleotide primed PCR (DOP-PCR). The approach can produce DNA libraries from nanograms of a template genome or the metagenome of multiple microbial populations. The DOP-PCR primers contain random bases, and thermodynamics of hairpin formation was used to design primers capable of binding randomly to template DNA for amplification with minimal bias. Next-generation high-throughput sequencing was used to determine the design is capable of amplifying up to 98% of template genomic DNA and consistently out-performed other DOP-PCR primers. Application of these new DOP-PCR amplified DNA libraries was demonstrated in multiple strain enrichments to isolate genetic library fragments capable of (i) increasing tolerance of E. coli ER2256 to toxic levels of 1-butanol by doubling the growth rate of the culture, (ii) redirecting metabolism to ethanol and pyruvate production (over 250% increase in yield) in Clostridium cellulolyticum when consuming cellobiose, and (iii) enhancing L-arginine production when used in conjunction with a new synthetic gene circuit.
Ph. D.
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9

Poblete, Castro Ignacio Andrés [Verfasser], and Christoph [Akademischer Betreuer] Wittmann. "Systems Biotechnology of Pseudomonas putida for the enhanced production of Polyhydroxyalkanoates: a rational approach for strain and bioprocess engineering / Ignacio Andrés Poblete Castro ; Betreuer: Christoph Wittmann." Braunschweig : Technische Universität Braunschweig, 2012. http://d-nb.info/1175823147/34.

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10

Montoya, Solano José David [Verfasser]. "Metabolic engineering of the Colombian strain Clostridium sp. IBUN 158B in order to improve the bioconversion of glycerol into 1,3-propanediol / José David Montoya Solano." Ulm : Universität Ulm. Fakultät für Naturwissenschaften, 2013. http://d-nb.info/1030045755/34.

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11

Lange, Anna [Verfasser], and Christoph [Akademischer Betreuer] Wittmann. "Bio-based production of succinate from renewable resources : elucidation of Basfia succiniciproducens metabolism by 13C metabolic flux analysis for knowledge-based strain engineering / Anna Lange ; Betreuer: Christoph Wittmann." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2017. http://d-nb.info/118298908X/34.

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12

Brown, Steven Richard. "A design of experiments approach for engineering carbon metabolism in the yeast Saccharomyces cerevisiae." Thesis, University of Exeter, 2016. http://hdl.handle.net/10871/26158.

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The proven ability to ferment Saccharomyces cerevisiae on a large scale presents an attractive target for producing chemicals and fuels from sustainable sources. Efficient and predominant carbon flux through to ethanol is a significant engineering issue in the development of this yeast as a multi-product cell chassis used in biorefineries. In order to evaluate diversion of carbon flux away from ethanol, combinatorial deletions were investigated in genes encoding the six isozymes of alcohol dehydrogenase (ADH), which catalyse the terminal step in ethanol production. The scarless, dominant and counter- selectable amdSYM gene deletion method was optimised for generation of a combinatorial ADH knockout library in an industrially relevant strain of S. cerevisiae. Current understanding of the individual ADH genes fails to fully evaluate genotype-by-genotype and genotype-by-environment interactions: rather, further research of such a complex biological process requires a multivariate mathematical modelling approach. Application of such an approach using the Design of Experiments (DoE) methodology is appraised here as essential for detailed empirical evaluation of complex systems. DoE provided empirical evidence that in S. cerevisiae: i) the ADH2 gene is not associated with producing ethanol under anaerobic culture conditions in combination with 25 g l-1 glucose substrate concentrations; ii) ADH4 is associated with increased ethanol production when the cell is confronted with a zinc-limited [1 μM] environment; and iii) ADH5 is linked with the production of ethanol, predominantly at pH 4.5. A successful metabolic engineering strategy is detailed which increases the product portfolio of S. cerevisiae, currently used for large-scale production of bioethanol. Heterologous expression of the cytochrome P450 fatty acid peroxygenase from Jeotgalicoccus sp., OleTJE, fused to the RhFRED reductase from Rhodococcus sp. NCIMB 978 converted free fatty acid precursors to C13, C15 and C17 alkenes (3.81 ng μl-1 total alkene concentration).
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13

Pechacek, Janet. "Metabolic and evolutionary engineering of a xylose-fermenting strain of Saccharomyces cerevisiae." Thesis, 2011. http://spectrum.library.concordia.ca/36124/1/Pechacek_MSc_2012.pdf.

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Lignocellulosic biomass waste is an abundant renewable resource of sugars for fermentation to biofuel. Due to its high fermentation capability and tolerance to ethanol and inhibitors, Saccharomyces cerevisiae was chosen to engineer a strain able to ferment xylose to ethanol. Wild-type S. cerevisiae is not able to grow on xylose as a sole carbon source. In xylose-fermenting yeasts, xylose reductase reduces the sugar to xylitol, which is then oxidized to xylulose by a xylitol dehydrogenase. These two enzymes require different cofactors, which leads to a cofactor imbalance in wild-type cells attempting to utilize xylose. In order to bypass the two-step oxidoreductive isomerization reaction, the xylose reductase-encoding GRE3 gene was knocked out and the pathway was replaced with a xylose isomerase (XYLA) isolated from Piromyces sp. E2, to convert xylose directly to xylulose. To increase the flux of xylulose towards the pentose phosphate pathway, a second copy of the endogenous xylulokinase (XKS1) was constitutively expressed. This two-gene construct was chromosomally integrated into the GRE3 deletion strains and the resulting strain was able to grow aerobically on xylose as its sole carbon source. Anaerobic glucose-xylose co-fermentation experiments yielded increased growth as compared to glucose only cultures, but ethanol production did not increase. In micro-aerobic high cell density fermentation the strains successfully produced ethanol from xylose as its sole carbon source and from a mixture of glucose and xylose. Evolutionary engineering further improved the growth rate, ethanol yield and specific ethanol productivity of these strains.
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14

Ranganathan, Sridhar Cavalier Tom M. "A study of optimization and algorithms for metabolic engineering and strain design." 2008. http://etda.libraries.psu.edu/theses/approved/WorldWideIndex/ETD-3186/index.html.

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15

Dahn, Kristine M. "Strain selection and metabolic engineering or Pichia stipitis for increased ethanol production from xylose." 1998. http://catalog.hathitrust.org/api/volumes/oclc/42360615.html.

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Thesis (Ph. D.)--University of Wisconsin--Madison, 1998.
Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 113-118).
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