Cane D E, Luo G, Khosla C, Kao C M, Katz L
Department of Chemistry, Brown University, Providence, Rhode Island 02912, USA.
J Antibiot (Tokyo). 1995 Jul;48(7):647-51. doi: 10.7164/antibiotics.48.647.
Feeding of (2S,3R)-[2,3-13C2]-2-methyl-3-hydroxypentanoyl NAC thioester (1a) to the recombinant organism Streptomyces coelicolor CH999/pCK7 harboring the complete set of eryA genes from Saccharopolyspora erythraea encoding the 6-deoxyerythronolide B synthase (DEBS) resulted in the formation of 6-deoxyerythronolide B (2a) labeled with 13C at C-12 and C-13, as evidenced by the appearance of a pair of enhanced and coupled doublets in the 13C NMR spectrum. The level of 13C enrichment was 15-20 atom% 13C, as much as 100 times higher than the usually observed efficiency of incorporation of NAC thioesters into polyketide metabolites. Similar incorporation of (2S,3R)-[3-2H,3-13C]-2-methyl-3-hydroxypentanoyl NAC thioester (1b) gave 6-deoxyerythronolide B (2b) labeled with both 13C and deuterium at C-13. The intact incorporation of both precursors confirms the normal functioning of the recombinant DEBS proteins in the heterologous host.
将（2S，3R）-[2,3-¹³C₂]-2-甲基-3-羟基戊酰N-乙酰半胱氨酸硫酯（1a）喂给含有来自糖多孢红霉菌的全套eryA基因的重组生物体天蓝色链霉菌CH999/pCK7，该基因编码6-脱氧红霉内酯B合酶（DEBS），结果形成了在C-12和C-13处标记有¹³C的6-脱氧红霉内酯B（2a），¹³C NMR谱中一对增强的耦合双峰的出现证明了这一点。¹³C富集水平为15-20原子％¹³C，比通常观察到的N-乙酰半胱氨酸硫酯掺入聚酮化合物代谢物的效率高100倍。（2S，3R）-[3-²H，3-¹³C]-2-甲基-3-羟基戊酰N-乙酰半胱氨酸硫酯（1b）的类似掺入产生了在C-13处同时标记有¹³C和氘的6-脱氧红霉内酯B（2b）。两种前体的完整掺入证实了重组DEBS蛋白在异源宿主中的正常功能。
