Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Hiroshima, Japan.
Hiroshima Research Center for Healthy Aging (HiHA), Hiroshima University, Hiroshima, Japan.
Biosci Biotechnol Biochem. 2021 Jan 7;85(1):115-125. doi: 10.1093/bbb/zbaa063.
To elucidate the gross lankamycin biosynthetic pathway including two cytochrome P450 monooxygenases, LkmK and LkmF, we constructed two double mutants of P450 genes in combination with glycosyltransferase genes, lkmL and lkmI. An aglycon 8,15-dideoxylankanolide, a possible substrate for LkmK, was prepared from an lkmK-lkmL double mutant, while a monoglycoside 3-O-l-arcanosyl-8-deoxylankanolide, a substrate for LkmF, was from an lkmF-lkmI double mutant. Bioconversion of lankamycin derivatives was performed in the Escherichia coli recombinant for LkmK and the Streptomyces lividans recombinant for LkmF, respectively. LkmK catalyzes the C-15 hydroxylation on all 15-deoxy derivatives, including 8,15-dideoxylankanolide (a possible substrate), 8,15-dideoxylankamycin, and 15-deoxylankamycin, suggesting the relaxed substrate specificity of LkmK. On the other hand, LkmF hydroxylates the C-8 methine of 3-O-l-anosyl-8-deoxylankanolide. Other 8-deoxy lankamycin/lankanolide derivatives were not oxidized, suggesting the importance of a C-3 l-arcanosyl moiety for substrate recognition by LkmF in lankamycin biosynthesis. Thus, LkmF has a strict substrate specificity in lankamycin biosynthesis.
为了阐明包括两个细胞色素 P450 单加氧酶 LkmK 和 LkmF 在内的大拉科霉素生物合成途径，我们构建了两个 P450 基因与糖基转移酶基因 lkmL 和 lkmI 的双突变体。一种可能是 LkmK 底物的无糖基 8,15-去氧拉科诺内酯，是从 lkmK-lkmL 双突变体中制备的，而一种单糖苷 3-O-l-岩藻糖基-8-去氧拉科诺内酯，是 LkmF 的底物，是从 lkmF-lkmI 双突变体中制备的。拉科霉素衍生物的生物转化分别在大肠杆菌重组体 LkmK 和链霉菌重组体 LkmF 中进行。LkmK 催化所有 15-脱氧衍生物的 C-15 羟化，包括 8,15-去氧拉科诺内酯（可能的底物）、8,15-去氧拉科霉素和 15-去氧拉科霉素，这表明 LkmK 的底物特异性较宽松。另一方面，LkmF 羟化 3-O-l-岩藻糖基-8-去氧拉科诺内酯的 C-8 亚甲基。其他 8-去氧拉科霉素/拉科诺内酯衍生物没有被氧化，这表明在拉科霉素生物合成中，C-3 l-岩藻糖基部分对于 LkmF 识别底物的重要性。因此，LkmF 在拉科霉素生物合成中具有严格的底物特异性。
