Metabolic model design and elementary mode analysis of Shewanella oneidensis MR-1 and Derivative Strains & Plasmid construction to facilitate PHB production in Saccharomyces cerevisiae using a single vector.
2010-04
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Metabolic model design and elementary mode analysis of Shewanella oneidensis MR-1 and Derivative Strains & Plasmid construction to facilitate PHB production in Saccharomyces cerevisiae using a single vector.
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2010-04
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In Chapter 1, a stoichiometric model describing the central metabolism of Shewanella oneidensis MR-1 wild-type and derivative strains was developed for use in elementary mode (EM) analysis. An EM model was created and verified by comparing growth phenotypes of single- and double-knockout strains cultivated on differing carbon sources, under aerobic and anaerobic conditions. Furthermore, several single knockout growth phenotypes under specific conditions have been predicted, offering potential for model verification. This wild-type model can be easily adapted to different carbon sources or metabolic products, and allows the prediction of single- and multiple- knockout strains that are expected to operate under defined conditions with increased efficiency when compared to wild type cells. To this end, the S. oneidensis wild-type EM model was adapted by addition of both glycerol import reactions and reactions needed to facilitate ethanol production from pyruvate. EM simulations were then performed on this glycerol-ethanol model to predict that the optimal anaerobic, ethanol producing strain cultivated on glycerol would contain the following mutations: Δpta ΔaldA ΔgcvT. This strain is expected to require ethanol secretion to accumulate biomass under anaerobic conditions, and as a result to produce ethanol yields between 0.4668 mol ethanol/mol glycerol and 1 mol ethanol/mol glycerol consumed (theoretical yield is 1 mol/mol). Future work should focus on performing these gene deletions and characterizing the resultant mutant strains, as well as determining the role of formate in anaerobic shewanella metabolism.
In addition, the steps in generating a single plasmid which expresses all three R. eutropha PHA synthesis genes in Saccharomyces cerevisisae is described in Chapter 2. Several methods were employed to attempt to generate this plasmid, while plasmid construction was successful using yeast-mediated ligation and inserting the TEF1-phaC1 promoter-gene into p2DPT-RK(U) to create the plasmid p2DPTT1-RKS1(U). The GAL1-10 promoter is present in the host vector, and it is likely that previous efforts were hampered by plasmid instability resulting from undesired intra-plasmid homologous recombination between these duplicate GAL1-10 promoter regions. The exact sequence of the region downstream of the phaC1 gene should be determined prior to application PHA production and other experiments. This plasmid should facilitate the direct observation of PHA accumulation in vivo using fluorescence microscopy, as well as improve PHA yields in transformed strains when compared to the two-plasmid system.
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University of Minnesota M.S. thesis. April 2010. Major: Microbial Engineering. Advisor: Friedrich Srienc. 1 computer file (PDF); vii, 93 pages. Ill. (some col.)
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Flynn, Chris M.. (2010). Metabolic model design and elementary mode analysis of Shewanella oneidensis MR-1 and Derivative Strains & Plasmid construction to facilitate PHB production in Saccharomyces cerevisiae using a single vector.. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/90881.
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