FIG. 2.

FK228 biosynthetic (dep) gene cluster and a proposed model for FK228 biosynthesis. (A) Physical map of clones and genes. pP4-G7 and pP4-B4 are positively identified genome sampling clones, and each contains part of the depD gene. The insert in pP4-G7 was labeled as a DNA probe to obtain cosmids 18 and 2. Cosmid 18 was shotgun sequenced. pCos2S1 to pCos2S5 are subclones of cosmid 2 and were sequenced by a primer walking method. Predicted genes in the dep gene cluster are designated depA to depN, and open reading frames outside the dep gene cluster are designated orf1 to orf3 and orf18 to orf21. Genes indicated by solid bars (depA to depJ) were predicted to be in the dep gene cluster with confidence; genes indicated by gray bars (depK to depN) were predicted to be in the dep gene cluster with less confidence. (B) Proposed model of FK288 biosynthesis by a hybrid NRPS-PKS-NRPS assembly line, including accessory activities of discrete proteins. PKS and NRPS domains are described in the text. A superscript “i” indicates that a domain is inactive; a superscript “n” indicates that a domain is nonfunctional. Inactive and nonfunctional domains are light gray. AL, acyl coenzyme A ligase; KS, β-ketoacyl synthase; E, epimerase.