High-Level Heterologous Production of <scp>d</scp> -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 &#177; 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 &#177; 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 H 2 S. 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 &#177; 57 μM, which is comparable to that of d -CS-producing S. lavendulae ATCC 11924 (930 &#177; 36 μM).