T he cyclic structural analog of the amino acid D-cycloserine (D-CS) ( Fig. 1) is a broad-spectrum antibiotic produced by Streptomyces lavendulae and Streptomyces garyphalus (1). The antibiotic inhibits both alanine racemase and D-alanyl-D-alanine ligase, which are necessary for the biosynthesis of peptidoglycan in the bacterial cell wall (2, 3). Rifampin and isoniazid have been clinically used for the treatment of tuberculosis caused by infection with Mycobacterium tuberculosis (4). However, M. tuberculosis that is resistant to these drugs has recently occurred. Presently, D-CS is clinically used as a second-line-of-defense drug against these antibiotic-resistant M. tuberculosis strains (4). In this connection, it has been shown that M. smegmatis overproducing alanine racemase is resistant to D-CS (5, 6). Recently, D-CS has been shown to function as a partial agonist for the N-methyl-D-aspartate receptor. As a result, the application of D-CS for the treatment of some psychological dysfunctions has been extensively studied (7-9).Our group has successfully cloned a D-CS biosynthetic gene cluster from the chromosomal DNA of D-CS-producing S. lavendulae ATCC 11924, which is composed of 10 open reading frames, designated dcsA to dcsJ (10). The functions of dcsI and dcsJ had previously been analyzed using the corresponding genes cloned from other D-CS-producing strains, i.e., S. lavendulae ATCC 25233 (11) and S. garyphalus (CSH) 5-12 (12), demonstrating that both gene products are responsible for self-resistance in the D-CS producer. Gene disruption and recombinant protein analyses have demonstrated that the revised D-CS biosynthetic pathway is as follows. L-Serine is O-acetylated by DcsE to generate O-acetyl-L-serine (OAS) (10, 13). The resultant OAS reacts with hydroxyurea (HU) to yield O-ureido-L-serine by use of DcsD, which is a pyridoxal phosphate-dependent enzyme (14). O-Ureido-Lserine is racemized by DcsC (14, 15), followed by cyclization with DcsG, which is a member of the ATP-grasp fold family of proteins (10, 14) (Fig. 1).We have previously hypothesized that L-arginine, as a precursor in the D-CS biosynthetic pathway, must be hydroxylated by nitric oxide synthase (NOS) expressed in D-CS-producing S. lavendulae (10). However, we have corrected the hypothesis as follows: DcsA as a heme protein, but not as an NOS protein, contributes to the formation of N -hydroxy-L-arginine (16). As shown in Fig. 1, HU is generated by the hydrolysis of N -hydroxy-L-arginine with DcsB (10).In recent years, the heterologous expression of secondary metabolic pathways using a surrogate host, such as Escherichia coli, has emerged as an effective way of producing natural products. However, practical antibiotics have not yet been successfully produced using E. coli as a host. Our goal is to realize high production of D-CS by expressing its biosynthetic genes (dcsA to -dcsE and dcsG) in E. coli as a host cell. In this study, we tried to introduce the four D-CS biosynthetic genes (dcsC, dcsD, dcsE, and dcsG) into E. coli cells to expr...