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/mdaat(+)) grew well when supplemented with certain D-amino acid. A multicopy suppression study with plasmids encoding one of the 51 PLPdependent enzymes of E. coli showed that MB3000/mdaat(+) could detect weak and moonlighting racemase activity, such from cystathionine beta-lyase (MetC) and a negative regulator of MalT activity/cystathionine beta-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.

Automated summary

In recent years, it has been found that most existing organisms do not depend on D-amino acids for growth, limiting our understanding of their biological functions. However, researchers have discovered an Escherichia coli strain lacking major D-Ala and D-Glu biosynthetic enzymes that can grow well when supplemented with certain D-amino acids. This finding provides a new platform for further research on D-amino acid-metabolizing enzymes.