Mendes M V, Recio E, Fouces R, Luiten R, Martín J F, Aparicio J F
Institute of Biotechnology INBIOTEC, Parque Científico de León, Spain.
Chem Biol. 2001 Jul;8(7):635-44. doi: 10.1016/s1074-5521(01)00033-3.
The post-polyketide synthase biosynthetic tailoring of polyene macrolides usually involves oxidations catalysed by cytochrome P450 monooxygenases (P450s). Although members from this class of enzymes are common in macrolide biosynthetic gene clusters, their specificities vary considerably toward the substrates utilised and the positions of the hydroxyl functions introduced. In addition, some of them may yield epoxide groups. Therefore, the identification of novel macrolide monooxygenases with activities toward alternative substrates, particularly epoxidases, is a fundamental aspect of the growing field of combinatorial biosynthesis. The specific alteration of these activities should constitute a further source of novel analogues. We investigated this possibility by directed inactivation of one of the P450s belonging to the biosynthetic gene cluster of an archetype polyene, pimaricin.
A recombinant mutant of the pimaricin-producing actinomycete Streptomyces natalensis produced a novel pimaricin derivative, 4,5-deepoxypimaricin, as a major product. This biologically active product resulted from the phage-mediated targeted disruption of the gene pimD, which encodes the cytochrome P450 epoxidase that converts deepoxypimaricin into pimaricin. The 4,5-deepoxypimaricin has been identified by mass spectrometry and nuclear magnetic resonance following high-performance liquid chromatography purification.
We have demonstrated that PimD is the epoxidase responsible for the conversion of 4,5-deepoxypimaricin to pimaricin in S. natalensis. The metabolite accumulated by the recombinant mutant, in which the epoxidase has been knocked out, constitutes the first designer polyene obtained by targeted manipulation of a polyene biosynthetic gene cluster. This novel epoxidase could prove to be valuable for the introduction of epoxy substituents into designer macrolides.
聚酮合酶后修饰的多烯大环内酯生物合成通常涉及细胞色素P450单加氧酶（P450s）催化的氧化反应。尽管这类酶的成员在大环内酯生物合成基因簇中很常见，但它们对所利用的底物以及引入的羟基功能位置的特异性差异很大。此外，其中一些酶可能会产生环氧基团。因此，鉴定对替代底物具有活性的新型大环内酯单加氧酶，特别是环氧化酶，是组合生物合成这一不断发展的领域的一个基本方面。这些活性的特异性改变应构成新型类似物的另一个来源。我们通过对属于原型多烯匹马霉素生物合成基因簇的一种P450进行定向失活来研究这种可能性。
产生匹马霉素的链霉菌纳塔尔链霉菌的重组突变体产生了一种新型匹马霉素衍生物4,5 - 去氧匹马霉素作为主要产物。这种生物活性产物是由噬菌体介导的对基因pimD的靶向破坏产生的，该基因编码将去氧匹马霉素转化为匹马霉素的细胞色素P450环氧化酶。经过高效液相色谱纯化后，通过质谱和核磁共振鉴定了4,5 - 去氧匹马霉素。
我们已经证明PimD是纳塔尔链霉菌中负责将4,5 - 去氧匹马霉素转化为匹马霉素的环氧化酶。重组突变体积累的代谢产物（其中环氧化酶已被敲除）构成了通过靶向操纵多烯生物合成基因簇获得的首个设计多烯。这种新型环氧化酶可能被证明对于将环氧取代基引入设计大环内酯中具有价值。
