Chemical structures of nikkomycin X and Z and strategy for reassembling the entire nikkomycin gene cluster in an integrative plasmid. (A) Chemical structures of nikkomycin X and Z. (B) Construction of the nikkomycin biosynthetic gene cluster. The nikkomycin biosynthetic gene cluster consists of 22 ORFs (sanG-sanX) and is located on a supercos 1 based cosmid cosG4 and a pSET152 based plasmid pLXP. cosG4 contains most of the biosynthetic genes ranging from 8 kb DNA fragment upstream of sanG to partial sanDE; pLXP contains several biosynthetic genes ranging from partial sanI to 3 kb downstream of sanX. A 6 kb overlapping fragment between cosG4 and pLXP could be used as the lower recombinant arm. The upper arm was generated by insertion of a 4.5 kb DNA fragment from the upstream region of sanG into the pLXP to give pLXP-NRPS. The resulting pLXP-NRPS was linearized and then used to reassemble the whole cluster by Red/ET recombination with cosG4. The final construct containing the complete nikkomycin biosynthetic gene cluster was designated pNIK.