Precursor-Directed Biosynthesis of 16-Membered Macrolides by the Erythromycin Polyketide Synthase

Streptomyces coelicolor CH999/pJRJ2 harbors a plasmid encoding DEBS(KS1°), 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 (2E,4S,5R)-2,4-dimethyl-5-hydroxy-2-heptenoic
acid, N-acetylcysteamine thioester (6) an unsaturated triketide analogue of the natural triketide chain elongation
intermediate to cultures of S. coelicolor CH999/pJRJ2 results in formation of a 16-membered macrolactone,
which is isolated in the hemiketal form 33. The formation of the octaketide 33 indicates that the triketide
substrate has been processed by DEBS module 2 as if it were a diketide analogue. The substrate specificity of
this novel reaction has been explored by the incubation of three additional analogues of the unsaturated triketide
6, compounds 18, 31, and 32, with S. coelicolor CH999/pJRJ2, resulting in the formation of the corresponding
macrolactones 34, 35, and 36. By contrast, the unsaturated triketide 10, lacking a methyl group at C-2, did not
give rise to any detectable macrolactone product when incubated with S. coelicolor CH999/pJRJ2.

天蓝色链霉菌（Streptomyces coelicolor）CH999/pJRJ2携带编码DEBS(KS1°)的质粒，该质粒表达的是6-脱氧红霉内酯B合酶（6-deoxyerythronolide B synthase）的突变体；由于酮合酶（ketosynthase，KS1）结构域的活性位点发生突变，该突变体无法合成6-脱氧红霉内酯B（1, 6-dEB）——该结构域本应催化6-dEB生物合成的第一步聚酮链延伸反应。向天蓝色链霉菌CH999/pJRJ2的培养物中添加(2E,4S,5R)-2,4-二甲基-5-羟基-2-庚烯酸-N-乙酰半胱胺硫酯（化合物6），该物质为天然三酮聚酮链延伸中间体的不饱和三酮聚酮类似物，可得到16元大环内酯产物，该产物以半缩酮形式（产物33）分离得到。八酮聚酮产物33的形成表明，该三酮底物被DEBS模块2识别处理的方式与二酮聚酮类似物一致。通过将另外三种不饱和三酮聚酮类似物（化合物18、31和32）与天蓝色链霉菌CH999/pJRJ2孵育，探究了该新型反应的底物特异性，最终得到了对应的大环内酯产物34、35和36。与之相反，C2位缺乏甲基的不饱和三酮聚酮10与天蓝色链霉菌CH999/pJRJ2孵育后，未产生任何可检测到的大环内酯产物。