Analysis of the loading and hydroxylation steps in lankamycin biosynthesis in Streptomyces rochei
Analysis of the loading and hydroxylation steps in lankamycin biosynthesis in Streptomyces rochei
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DOI：
10.1128/aac.00016-06
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发表时间：
2006-06-01
影响因子：
4.9
通讯作者：
Kinashi, Haruyasu
中科院分区：
文献类型：
--
作者：
Arakawa, Kenji;Kodama, Kazuya;Kinashi, Haruyasu
兰卡霉素（LM）是一种14元大环内酯类抗生素，其生物合成基因簇编码于罗氏链霉菌7434 AN 4的210-kb线性质粒pSLA 2-L上。LM在C-13位含有3-羟基-2-丁基，其不同于红霉素中的乙基。对于LM生物合成的起始部分的起源，可以考虑以下两种可能性：（i）额外的模块存在于生物合成基因簇中并加载额外的乙酸分子，或（ii）3-羟基-2-丁酸或其等同物被加载并作为起始物掺入。通过pSLA 2-L的完全测序排除了前一种可能性，其显示没有额外的模块。另一方面，后者通过将[3-H-2] DL-异亮氨酸中的氘掺入LM的C-14位来证实。通过构建两个P450羟化酶基因lkmF（orf 26）和lkmK（orf 37）的破坏体，研究了LM的C-15和C-8位的羟化反应的时间。lkmF破坏物产生8-去氧代坎霉素，而lkmK破坏物产生15-去氧代坎霉素和8，15-双去氧代坎霉素。这些结果清楚地表明，在LM生物合成中，LkmF是C-8羟化酶，LkmK是C-15羟化酶，此外还表明了羟基化步骤的顺序;即，羟基化可能首先在C-15处被LkmK发生，然后在C-8处被LkmF发生。
The biosynthetic gene cluster of lankamycin (LM), a 14-member macrolide antibiotic, is encoded on the 210-kb linear plasmid pSLA2-L in Streptomyces rochei 7434AN4. LM contains a 3-hydroxy-2-butyl group at the C-13 position, which is different from an ethyl group in erythromycin. The following two possibilities could be considered for the origin of this starter moiety of LM biosynthesis: (i) an extra module exists in the biosynthetic gene cluster and loads an additional acetate molecule, or (ii) 3-hydroxy-2-butyrate or its equivalent is loaded and incorporated as a starter. The former possibility was eliminated by the complete sequencing of pSLA2-L, which showed no extra module. On the other hand, the latter was confirmed by incorporation of deuterium in [3-H-2]DL-isoleucine into the C-14 position of LM. The timing of hydroxylation reactions at the C-15 and C-8 positions of LM was studied by constructing disruptants of two P450 hydroxylase genes, lkmF (orf26) and lkmK (orf37). The lkmF disruptant produced 8-deoxylankamycin, while the lkmK disruptant produced both 15-deoxylankamycin and 8,15-dideoxylankamycin. These results clearly showed that LkmF is a C-8 hydroxylase and LkmK is a C-15 hydroxylase in LM biosynthesis and in addition suggested the order of hydroxylation steps; namely, hydroxylation may occur at first at C-15 by LkmK and then at C-8 by LkmF.