Porting the synthetic D-glucaric acid pathway fromEscherichia colito Saccharomyces cerevisiae
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BTJ_submitted_05Jun2016.pdf
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9.38 MB
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Author(s)
Gupta, Amita
Hicks, Michael A.
Manchester, Shawn
Prather, Kristala L
Date Issued
September 2016
Journal
Biotechnology Journal
Publisher
Wiley
Citation
Gupta, Amita et al. "Porting the synthetic D‐glucaric acid pathway from Escherichia coli to Saccharomyces cerevisiae." Biotechnology Journal 11, 9 (September 2016): 1201-1208 © 2016 Wiley
Version
Original manuscript
Abstract
D-Glucaric acid can be produced as a value-added chemical from biomass through a de novo pathway in Escherichia coli. However, previous studies have identified pH-mediated toxicity at product concentrations of 5 g/L and have also found the eukaryotic myo-inositol oxygenase (MIOX) enzyme to be rate-limiting. We ported this pathway to Saccaromyces cerevisiae, which is naturally acid-tolerant and evaluate a codon-optimized MIOX homologue. We constructed two engineered yeast strains that were distinguished solely by their MIOX gene – either the previous version from Mus musculus or a homologue from Arabidopsis thaliana codon-optimized for expression in S. cerevisiae – in order to identify the rate-limiting steps for D-glucaric acid production both from a fermentative and non-fermentative carbon source. myo-Inositol availability was found to be rate-limiting from glucose in both strains and demonstrated to be dependent on growth rate, whereas the previously used M. musculus MIOX activity was found to be rate-limiting from glycerol. Maximum titers were 0.56 g/L from glucose in batch mode, 0.98 g/L from glucose in fed-batch mode, and 1.6 g/L from glucose supplemented with myo-inositol. Future work focusing on the MIOX enzyme, the interplay between growth and production modes, and promoting aerobic respiration should further improve this pathway. Keywords: Biochemical engineering; Bioprocess development; D-glucaric acid; Myo-inositol; Yeast
MIT Department
Massachusetts Institute of Technology. Department of Chemical Engineering
Massachusetts Institute of Technology. Synthetic Biology Center
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Creative Commons Attribution-Noncommercial-Share Alike
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DOI of Published Version
https://doi.org/10.1002/biot.201500563
