ITERATIVE TYPE-II POLYKETIDE SYNTHASES, CYCLASES AND KETOREDUCTASES EXHIBIT CONTEXT-DEPENDENT BEHAVIOR IN THE BIOSYNTHESIS OF LINEAR AND ANGULAR DECAPOLYKETIDES

Background: Iterative type II polyketide synthases (PKSs) produce poly ketide chains of variable but defined length from a specific starter u nit and a number of extender units. They also specify the initial regi ospecific folding and cyclization pattern of nascent polyketides eithe r through the action of a cyclase (CYC) subunit or through the combine d action of site-specific ketoreductase (KR) and CYC subunits. Additio nal CYCs and other modifications may be necessary to produce linear ar omatic polyketides. The principles of the assembly of the linear aroma tic polyketides, several of which are medically important, are well un derstood, but it is not clear whether the assembly of the angular arom atic (angucyclic) polyketides follows the same rules. Results: We perf ormed an in vivo evaluation of the subunits of the PKS responsible for the production of the angucyclic polyketide jadomycin (jad), in compa rison with their counterparts from the daunorubicin (dps) and tetracen omycin (tom) PKSs which produce linear aromatic polyketides. No matter which minimal PKS was used to produce the initial polyketide chain, t he JadD and DpsF CYCs produced the same two polyketides, in the same r atio; neither product was angularly fused. The set of jadABCED PKS plu s putative jadl CYC genes behaved similarly. Furthermore, no angular p olyketides were isolated when the entire set of jad PKS enzymes and Ja dl or the jad minimal PKS, Jadl and the TcmN CYC were present. The Dps E KR was able to reduce decaketides but not octaketides; in contrast, the KRs from the jad PKS (JadE) or the actinorhodin PKS (ActIII) could reduce octaketide chains, giving three distinct products. Conclusions : It appears that the biosynthesis of angucyclic polyketides cannot be simply accomplished by expressing the known PKS subunits from artific ial gene cassettes under the control of a non-native promoter. The cha racteristic structure of the angucycline ring system may arise from a kinked precursor during later cyclization reactions involving addition al, but so far unknown, components of the extended decaketide PKS. Our results also suggest that some KRs have a minimal chain length requir ement and that: CYC enzymes may act aberrantly as first-ring aromatase s that are unable to perform all of the sequential cyclization steps. Both of these characteristics may limit the widespread application of CYC or KR enzymes in the synthesis of novel polyketides.