Pathway and protein engineering for improved glucaric acid production in Escherichia coli
Author(s)Guay, Lisa Marie,Ph. D.Massachusetts Institute of Technology.
Massachusetts Institute of Technology. Department of Chemical Engineering.
Kristala L. J. Prather.
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Microbial fermentation is an attractive method for the renewable production of chemicals. Glucaric acid was identified as a "top value added chemical from biomass" by the Department of Energy in 2004, and a biological route for its production from glucose in E. coli was developed in our lab in 2009. Two of the pathway enzymes, myo-inositol phosphate synthase (MIPS) and myoinositol oxygenase (MIOX), appear to control flux. This work addressed several limitations of these reactions. One approach was the relief of reactive oxygen species (ROS) to improve MIOX performance. MIOX converts myo-inositol (MI) to glucuronic acid. Overexpression of native catalase and superoxide dismutases led to significantly higher titers of glucuronic acid from MI. This result corresponded to better maintenance of MIOX activity and expression over the course of the fermentation. A reduction in labile iron levels, which are linked to ROS formation, was also shown to improve glucuronic acid titers.A second approach was the examination of natural MIPS diversity. MIPS competes with central carbon metabolism for its substrate, glucose-6-phosphate. Thirty-one representative MIPS homologs were selected using a sequence similarity network. Nineteen variants produced detectible myo-inositol (MI) from glucose, and H. contortus MIPS performed equally well or better than the current S. cerevisiae MIPS. Interesting differences in stability were identified between the variants, and further work to explore the network may yield more information about important sequence features. A third approach was the evaluation of screening methods for glucuronic and glucaric acid to support protein engineering. We attempted to extend a previous screen to growth from glucose, but while growth was achieved from MI, low flux appeared to prevent growth from glucose. A previously-developed biosensor based on the regulator CdaR was also tested.We discovered that the biosensor does not respond to glucaric acid but instead to a downstream metabolite, likely glycerate, and that the biosensor is affected by catabolite repression. While a reliable screen was not realized, our improved understanding of native regulation aids in the identification of alternative strategies. This work overall produced significant improvements in the glucaric acid pathway and helped to identify opportunities for further development.
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2019Cataloged from PDF version of thesis.Includes bibliographical references (pages 115-124).
DepartmentMassachusetts Institute of Technology. Department of Chemical Engineering
Massachusetts Institute of Technology