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

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/m daat + ) 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/m daat + 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.