Precursor-directed biosynthesis:: Stereospecificity for branched-chain diketides of the β-ketoacyl-ACP synthase domain 2 of 6-deoxyerythronolide B synthase

Modular polyketide synthases such as 6-deoxyerythronolide B synthase (DEBS) catalyze the biosynthesis of structurally complex natural products. Streptomyces coelicolor CH999/pJRJ2 harbors a plasmid encoding DEBS(KS1(0)), a mutant form of 6-deoxyerythronolide B synthase that is blocked in the formation of 6-deoxyerythronolide B (1, 6-dEB) due to a mutation in the active site of the ketosynthase (KS1) domain that normally catalyzes the first polyketide chain-elongation step of 6-dEB biosynthesis. Administration of (2S,3R,4S)- and (2S,3R,4R)-3-hydroxy-2,4-dimethylhexanoic acid N-acetylcysteamine (SNAC) thioesters (= S-[2-(acetylamino)ethyl] (2S,3R,4S)- and (2S,3R,4R)-3-hydroxy-2,4-dimethylhexanethioates) 3 and 4 in separate experiments to cultures of Streptomyces coelicolor CH999/pJRJ2 led to production of the corresponding (14S)- and (14R)-14-methyl analogues of 6-dEB, 10 and 11, respectively. Unexpectedly, when a 3:2 mixture of 4 and 3 was fed under the same conditions, exclusively branched-chain macrolactone 11 was isolated. In similar experiments, feeding of 3 and 4 to S. coelicolor CH999/pCK16, an engineered strain harboring DEBS1 + TE(KS1(0)), resulted in formation of the branched-chain triketide lactones 13 and 14, while feeding of the 3:2 mixture of 4 and 3 gave exclusively 14. The biochemical basis for this stereochemical discrimination was established by using purified DEBS module 2 + TE to determine the steady-state kinetic parameters for 3 and 4, with the k(cat)/K-M for 4 shown to be sevenfold greater than that of 3.