ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES FROM STREPTOMYCES SPORE PIGMENT POLYKETIDE SYNTHASES

A series of 12 recombinants expressing sets of polyketide synthase (PK S) genes from the whiE (Streptomyces coelicolor), sch (S. halstedii), and cur (S. curacoi) spore pigment biosynthetic gene Clusters were pre pared and shown to produce four groups of novel polyketides. Mixtures of undecaketides and dodecaketides were produced by the minimal PKS al one (TW93b, TW93c, and TW93d) or in the presence of the (unnatural) ac t ketoreductase (KR) (TW94b, TW94c, and TW94d), whereas when the whiE- ORFVI cyclase was present, only dodecaketides (TW95a and TW95b) arose, in high yield. This implies that the whiE minimal PKS requires an add itional subunit (the cyclase) to stabilize the complex between the lon g nascent polyketide chain and the minimal PKS to ensure that the chai n reaches the full 24 carbons. These experiments suggest that the nati ve spore pigment is a C24 molecule with a pentacenequinone structure w hich is first cyclized C9 to C14. A fourth set of uncharacterized poly ketides was produced when the complete set of three WhiE cyclases was expressed together with the whiE minimal PKS. It seems that the cyclas es, the products of whiE-ORFs II and VII, act in concert with the rema inder of the whiE PKS subunits to facilitate construction of the nearl y complete spore pigment polyketide. Shortened polyketides were additi onally produced by the minimal PKS alone (the heptaketide TW93a) and i n the presence of the act KR (the pentaketide orcacetophenone, TW93a). While these polyketides might be derailment products resulting from a promiscuous chain length factor, they could also arise as degradation products from intermediates in the biosynthesis of the structurally r elated larger polyketides. Finally, the isolation of the same aromatic polyketide products from the recombinants carrying corresponding gene s from the whiE, sch, and cup gene clusters suggests that the various spore pigments observed in Streptomyces spp. are derived from similar or identical polycyclic aromatic polyketide intermediates.