Metabolic engineering of microorganisms for the overproduction of fatty acids

dc.contributor.advisor Jacqueline V. Shanks Tee, Ting Wei
dc.contributor.department Chemical and Biological Engineering 2018-07-21T16:01:34.000 2020-06-30T02:50:16Z 2020-06-30T02:50:16Z Tue Jan 01 00:00:00 UTC 2013 2015-07-30 2013-01-01
dc.description.abstract <p>Fatty acids naturally synthesized in many organisms are promising starting points for the catalytic production of industrial chemicals and diesel-like biofuels. However, bio-production of fatty acids in microbial hosts relies heavily on manipulating tightly regulated fatty acid biosynthetic pathways, thus complicating the engineering for higher yields. With the advent of systems metabolic engineering, we demonstrated an iterative metabolic engineering effort that integrates computationally driven predictions and metabolic flux analysis (MFA) was demonstrated to meet this challenge. With wild type E. coli fluxomic data, the OptForce procedure was employed to suggest genetic manipulations for fatty acid overproduction. In accordance with the OptForce prioritization of interventions, fabZ and acyl-ACP thioesterase were upregulated and fadD was deleted to arrive at a strain that produces 1.70 g/L and 0.14 g fatty acid/g glucose of C14-16 fatty acid in minimal medium. However, OptForce does not infer gene regulation, enzyme inhibition and metabolic toxicity. Along with transcriptomics and metabolomics analysis, we re-deployed OptForce simulation using the redefined flux distribution as constraints to generate predictions for the second generation fatty acid-overproducing strain. MFA identified the up-regulation of the TCA cycle and down-regulation of pentose phosphate pathway under fatty acid overproduction to replenish the need of energy and reducing molecules. The elevation of intracellular metabolite levels in the TCA cycle complemented the flux findings. With re-defined flux boundary of the first generation strain, OptForce suggested the interruption of TCA cycle such as removal of succinate dehydrogenase as the most prioritized genetic intervention to further improve fatty acid production. Meanwhilem, the whole genome transcriptional analysis revealed acid stress response, membrane disruption, colanic acid and biofilm formation during fatty acid production, thus pinpointing the targets for future metabolic engineering effort. These results highlight the benefit of using computational strain design and system metabolic engineering tools in systematically guiding the strain design to produce free fatty acids. Nonetheless, Saccharomyces cerevisiae is another attractive host organism for the production of biochemicals and biofuels. However, S. cerevisiae is very susceptible to octanoic acid toxicity. Transcriptomics analysis revealed membrane stress and intracellular acidification during octanoic acid stress. MFA illustrated the increase of flux in the TCA cycle possibly to facilitate the ATP-binding-cassette transporter activities. Further efforts can focus on improving membrane integrity or explore oleaginious yeasts to enhance the tolerance against fatty acids.</p>
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dc.identifier archive/
dc.identifier.articleid 4523
dc.identifier.contextkey 5050356
dc.identifier.s3bucket isulib-bepress-aws-west
dc.identifier.submissionpath etd/13516
dc.language.iso en
dc.source.bitstream archive/|||Fri Jan 14 19:54:45 UTC 2022
dc.subject.disciplines Chemical Engineering
dc.title Metabolic engineering of microorganisms for the overproduction of fatty acids
dc.type article
dc.type.genre dissertation
dspace.entity.type Publication
relation.isOrgUnitOfPublication 86545861-382c-4c15-8c52-eb8e9afe6b75 dissertation Doctor of Philosophy
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