Improving d-glucaric acid production from myo-inositol in E. coli by increasing MIOX stability and myo-inositol transport |
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Institution: | 1. Institute of Materia Medica, Chinese Academy of Medical Sciences, Peking Union Medical College, Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Beijing 100050, People''s Republic of China;2. Department of Pharmacy, The Second Affiliated Hospital of Nanchang, Nanchang 330000, People''s Republic of China |
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Abstract: | d-glucaric acid has been explored for a myriad of potential uses, including biopolymer production and cancer treatment. A biosynthetic route to produce d-glucaric acid from glucose has been constructed in Escherichia coli (Moon et al., 2009b), and analysis of the pathway revealed myo-inositol oxygenase (MIOX) to be the least active enzyme. To increase pathway productivity, we explored protein fusion tags for increased MIOX solubility and directed evolution for increased MIOX activity. An N-terminal SUMO fusion to MIOX resulted in a 75% increase in d-glucaric acid production from myo-inositol. While our directed evolution efforts did not yield an improved MIOX variant, our screen isolated a 941 bp DNA fragment whose expression led to increased myo-inositol transport and a 65% increase in d-glucaric acid production from myo-inositol. Overall, we report the production of up to 4.85 g/L of d-glucaric acid from 10.8 g/L myo-inositol in recombinant E. coli. |
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Keywords: | Soluble protein fusions Directed evolution Substrate transport Metabolic engineering |
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