d‐amino acid auxotrophic Escherichia coli strain for in vivo functional cloning of novel d‐amino acid synthetic enzyme

Abstract

Various d‐amino acids have been found in a wide range of organisms, including mammals. Although the physiological functions of various d‐amino acids have been reported or suggested, the molecular basis of these biological functions has been elucidated in only a few cases. The identification of a d‐amino acid biosynthetic enzyme is a critical step in understanding the mechanism of the physiological functions of d‐amino acids. While in vivo functional screening can be a powerful tool for identifying novel metabolic enzymes, none of the existing organisms exhibit growth dependent on d‐amino acid other than d‐Ala and d‐Glu. Here, we report the first organism that exhibits non‐canonical d‐amino acid auxotrophy. We found that an Escherichia coli strain lacking the major d‐Ala and d‐Glu biosynthetic enzymes, alr, dadX, and murI, and expressing the mutated d‐amino acid transaminase (DAAT) gene from Bacillus sp. YM‐1 (MB3000/mdaat+) grew well when supplemented with certain d‐amino acid. A multicopy suppression study with plasmids encoding one of the 51 PLP‐dependent enzymes of E. coli showed that MB3000/mdaat+ could detect weak and moonlighting racemase activity, such from cystathionine β‐lyase (MetC) and a negative regulator of MalT activity/cystathionine β‐lyase (MalY)—these exhibit only a few tenths to a few thousandths of the racemization activity of canonical amino acid racemases. We believe that this unique platform will contribute to further research in this field by identifying novel d‐amino acid‐metabolizing enzymes.