3 years ago

Production of ophthalmic acid using engineered Escherichia coli.

Ran Hori, Hisashi Hemmi, Tomokazu Ito, Tohru Yoshimura, Maiko Tokoro
Ophthalmic acid (l-γ-glutamyl-l-2-aminobutyryl-glycine, OA) is an analog of glutathione (l-γ-glutamyl-l-cysteinyl-glycine, GSH) in which the cysteine moiety is replaced by l-2-aminobutyrate. OA is a useful peptide for the pharmaceutical and/or food industries. Herein, we report a method for the production of OA using engineered Escherichia coli cells. The yggS-deficient E. coli cells, which lack the highly conserved pyridoxal 5' -phosphate-binding protein YggS and naturally accumulate OA, were selected as the starting strain background. To increase the production of OA, we overexpressed the OA biosynthetic enzymes glutamate-cysteine ligase (GshA) and glutathione synthase (GshB), desensitized the product inhibition of GshA, and eliminated the OA catabolic enzyme γ-glutamyltranspeptidase. The production of OA was further enhanced by the deletions of miaA and ridA with the aims of increasing the availability of ATP and attenuating the unwanted degradation of amino acids, respectively. The final strain developed in this study successfully produced 277 μmol/L of OA in 24 h without the formation of byproducts in a minimal synthetic medium containing 1 mM each of glutamate, 2-aminobutyrate, and glycine.IMPORTANCE Ophthalmic acid (OA) is a peptide that has the potential for use in the pharmaceutical and/or food industries. An efficient method for the production of OA would expand our knowledge about its physiological functions and enable the industrial/pharmaceutical application of this compound. We demonstrated the production of OA using Escherichia coli cells in which the OA biosynthetic and degradation enzymes have been engineered. We also showed that unique approaches including the use of ΔyggS as a starting strain, the establishment of an S495F mutation in GshA, and the deletion of ridA or miaA, facilitated the efficient production of OA in E. coli.

Publisher URL: http://doi.org/10.1128/AEM.02806-17

DOI: 10.1128/AEM.02806-17

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