High-level heterologous production of D-cycloserine by Escherichia coli

Abstract

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).