SPONTANEOUS PRIMING OF A DOWNSTREAM MODULE IN 6-DEOXYERYTHRONOLIDE-B SYNTHASE LEADS TO POLYKETIDE BIOSYNTHESIS

Modular polyketide synthases such as 6-deoxyerythronolide B synthase ( DEBS) catalyze the biosynthesis of structurally complex natural produc ts by repetitive condensation of simple carboxylic acid monomers. The synthase can be divided into groups of domains, called ''modules'', ea ch of which is responsible for one cycle of chain extension and proces sing The modular nature of these enzymes suggests that the biosyntheti c pathway might be rationally reprogrammed by manipulation of synthase s at the domain level. Although, several examples of successful engine ering of DEBS have been reported, a critical issue which has not been well-studied is the tolerance of ''downstream'' active sites to nonnat ural substrates. Here, we report that the terminal modules of DEBS, wh ich normally process highly functionalized intermediates, are competen t to carry out their natural functions on smaller, more simple substra tes. Expressed in the absence of other DEBS proteins, the DEBS3 protei n, which normally carries out the final two extension cycles in the sy nthesis of 6-deoxyerythronolide B (6-dEB), is spontaneously primed wit h a C-3 carboxylic acid. This substrate is then extended through two c ondensation cycles to form a triketide. Tolerance of the ''shortened'' intermediates in the biosynthesis of this triketide, in combination w ith results reported elsewhere [Jacobsen, J. R., Hutchinson, C. R., Ca ne, D. E., and Khosla, C. (1997) Science 277, 367-369], suggests that relaxed substrate specificity may be a common feature of modular polyk etide synthases. Interestingly, priming of DEBS3 appears to proceed, n ot by acyltransfer from propionyl-CoA, but by decarboxylation of an en zyme-bound methylmalonyl extender unit. This is the second example of decarboxylative priming within DEBS [see also Pieper, R., Gokhale, R. S., Luo, G., Cane, D. E., and Khosla, C. (1997) Biochemistry, 36, 1846 -1851] and suggests that, in the absence of an acceptable primer (or t ransferred intermediate), decarboxylative priming of ketosynthase doma ins may be a general property of modular polyketide synthases.