High-Level Heterologous Production of d -Cycloserine by Escherichia coli

Previously, we successfully cloned ad -cycloserine (d -CS) biosynthetic gene cluster consisting of 10 open reading frames (designateddcsA todcsJ ) fromd -CS-producingStreptomyces lavendulae ATCC 11924. In this study, we put fourd -CS biosynthetic genes (dcsC ,dcsD ,dcsE , anddcsG ) in tandem under the control of the T7 promoter in anEscherichia coli host. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. Whenl -serine and hydroxyurea (HU), the precursors ofd -CS, were incubated together with theE. coli resting cell suspension, the cells produced significant amounts ofd -CS (350 ± 20 μM). To increase the productivity ofd -CS, thedcsJ gene, which might be responsible for thed -CS excretion, was connected downstream of the four genes. TheE. coli resting cells harboring the five genes producedd -CS at 660 ± 31 μM. ThedcsD gene product, DcsD, formsO -ureido-l -serine fromO -acetyl-l -serine (OAS) and HU, which are intermediates ind -CS biosynthesis. DcsD also catalyzes the formation ofl -cysteine from OAS and H2 S. To repress the side catalytic activity of DcsD, theE. coli chromosomalcysJ andcysK genes, encoding the sulfite reductase α subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the fourd -CS biosynthetic genes, together withdcsJ , were incubated withl -serine and HU, thed -CS production was 980 ± 57 μM, which is comparable to that ofd -CS-producingS. lavendulae ATCC 11924 (930 ± 36 μM).