High-Level Heterologous Production of D-Cycloserine by Escherichia coli.
Previously, we successfully cloned a d-cycloserine (d-CS) biosynthetic gene cluster consisting of 10 open reading frames (designated dcsA to dcsJ) from d-CS-producing Streptomyces lavendulae ATCC 11924. In this study, we put four d-CS biosynthetic genes (dcsC, dcsD, dcsE, and dcsG) in tandem under the control of the T7 promoter in an Escherichia coli host. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. When l-serine and hydroxyurea (HU), the precursors of d-CS, were incubated together with the E. coli resting cell suspension, the cells produced significant amounts of d-CS (350 ± 20 μM). To increase the productivity of d-CS, the dcsJ gene, which might be responsible for the d-CS excretion, was connected downstream of the four genes. The E. coli resting cells harboring the five genes produced d-CS at 660 ± 31 μM. The dcsD gene product, DcsD, forms O-ureido-l-serine from O-acetyl-l-serine (OAS) and HU, which are intermediates in d-CS biosynthesis. DcsD also catalyzes the formation of l-cysteine from OAS and H2S. To repress the side catalytic activity of DcsD, the E. coli chromosomal cysJ and cysK genes, encoding the sulfite reductase α subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the four d-CS biosynthetic genes, together with dcsJ, were incubated with l-serine and HU, the d-CS production was 980 ± 57 μM, which is comparable to that of d-CS-producing S. lavendulae ATCC 11924 (930 ± 36 μM).
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Takanori
T
Kumagai
Department of Pharmaceutical Sciences, Hiroshima University, Hiroshima, Japan.
Tomoki
T
Ozawa
Department of Pharmaceutical Sciences, Hiroshima University, Hiroshima, Japan.
Momoko
M
Tanimoto
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Masafumi
M
Noda
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Yasuyuki
Y
Matoba
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan sugi@hiroshima-u.ac.jp.
Masanori
M
Sugiyama
D000890:Anti-Infective Agents / Q000378:metabolism*; D003523:Cycloserine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005810:Multigene Family; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000378:metabolism
D000890:Anti-Infective Agents; D003523:Cycloserine