High-Level Heterologous Production of d-Cycloserine by Escherichia coli
ABSTRACTPreviously, we successfully cloned ad-cycloserine (d-CS) biosynthetic gene cluster consisting of 10 open reading frames (designateddcsAtodcsJ) fromd-CS-producingStreptomyces lavendulaeATCC 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 colihost. 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. coliresting cell suspension, the cells produced significant amounts ofd-CS (350 ± 20 μM). To increase the productivity ofd-CS, thedcsJgene, which might be responsible for thed-CS excretion, was connected downstream of the four genes. TheE. coliresting cells harboring the five genes producedd-CS at 660 ± 31 μM. ThedcsDgene 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 H2S. To repress the side catalytic activity of DcsD, theE. colichromosomalcysJandcysKgenes, 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. lavendulaeATCC 11924 (930 ± 36 μM).