Laboratory of Microbial Engineering, Kitasato Institute for Life Sciences, Kitasato University, Minami-ku, Sagamihara, Kanagawa, Japan.
J Antibiot (Tokyo). 2011 Jan;64(1):65-71. doi: 10.1038/ja.2010.135. Epub 2010 Nov 17.
Pentalenic acid (1) has been isolated from many Streptomyces sp. as a co-metabolite of the sesquiterpenoid antibiotic pentalenolactone and related natural products. We have previously reported the identification of a 13.4-kb gene cluster in the genome of Streptomyces avermitilis implicated in the biosynthesis of the pentalenolactone family of metabolites consisting of 13 open reading frames. Detailed molecular genetic and biochemical studies have revealed that at least seven genes are involved in the biosynthesis of the newly discovered metabolites, neopentalenoketolactone, but no gene specifically dedicated to the formation of pentalenic acid (1) was evident in the same gene cluster. The wild-type strain of S. avermitilis, as well as its derivatives, mainly produce pentalenic acid (1), together with neopentalenoketolactone (9). Disruption of the sav7469 gene encoding a cytochrome P450 (CYP105D7), members of which class are associated with the hydroxylation of many structurally different compounds, abolished the production of pentalenic acid (1). The sav7469-deletion mutant derived from SUKA11 carrying pKU462∷ptl-clusterΔptlH accumulated 1-deoxypentalenic acid (5), but not pentalenic acid (1). Reintroduction of an extra-copy of the sav7469 gene to SUKA11 Δsav7469 carrying pKU462∷ptl-clusterΔptlH restored the production of pentalenic acid (1). Recombinant CYP105D7 prepared from Escherichia coli catalyzed the oxidative conversion of 1-deoxypentalenic acid (5) to pentalenic acid (1) in the presence of the electron-transport partners, ferredoxin (Fdx) and Fdx reductase, both in vivo and in vitro. These results unambiguously demonstrate that CYP105D7 is responsible for the conversion of 1-deoxypentalenic acid (5) to pentalenic acid (1), a shunt product in the biosynthesis of the pentalenolactone family of metabolites.
戊烯酸（1）已从许多链霉菌属中分离出来，作为倍半萜烯抗生素戊烯内酯和相关天然产物的共代谢物。我们之前曾报道过链霉菌属avermitilis 基因组中一个 13.4kb 的基因簇的鉴定，该基因簇参与戊烯内酯类代谢物的生物合成，由 13 个开放阅读框组成。详细的分子遗传和生化研究表明，至少有 7 个基因参与了新发现的代谢物，即 neo-pentalenoketolactone 的生物合成，但在同一基因簇中没有明显的专门用于形成戊烯酸（1）的基因。野生型链霉菌属 avermitilis 及其衍生物主要产生戊烯酸（1）和 neo-pentalenoketolactone（9）。编码细胞色素 P450（CYP105D7）的 sav7469 基因的破坏，该酶属于能够使许多结构不同的化合物羟化的同工酶家族，导致戊烯酸（1）的产量减少。来自 SUKA11 的 sav7469 缺失突变体携带 pKU462∷ptl-clusterΔptlH 积累了 1-脱氧戊烯酸（5），但没有戊烯酸（1）。将 sav7469 基因的额外拷贝重新引入到携带 pKU462∷ptl-clusterΔptlH 的 SUKA11 Δsav7469 中，恢复了戊烯酸（1）的产生。从大肠杆菌中制备的重组 CYP105D7 在电子传递伴侣铁氧还蛋白（Fdx）和 Fdx 还原酶存在下，无论是在体内还是在体外，都能催化 1-脱氧戊烯酸（5）氧化转化为戊烯酸（1）。这些结果明确表明，CYP105D7 负责将 1-脱氧戊烯酸（5）转化为戊烯酸（1），这是戊烯内酯类代谢物生物合成中的一个支路产物。
