Reconstitution of the FK228 Biosynthetic Pathway Reveals Cross Talk between Modular Polyketide Synthases and Fatty Acid Synthase
Reconstitution of the FK228 Biosynthetic Pathway Reveals Cross Talk between Modular Polyketide Synthases and Fatty Acid Synthase
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DOI：
10.1128/aem.01513-10
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发表时间：
2011-02-01
影响因子：
4.4
通讯作者：
Cheng, Yi-Qiang
中科院分区：
文献类型：
--
作者：
Wesener, Shane R.;Potharla, Vishwakanth Y.;Cheng, Yi-Qiang
在微生物中，脂肪酸生物合成和次级代谢之间的功能性串扰已在数个案例中被发现。然而，它们都不涉及模块化生物合成酶。此前，我们报道了一种混合模块化非核糖体肽合成酶（NRPS）-聚酮合酶（PKS）途径，用于在紫色色杆菌菌株968中生物合成FK228抗癌缩酚酸肽。该途径在DepBC酶上包含两个PKS模块，缺乏功能性酰基转移酶（AT）结构域，并且在基因簇或其附近不存在明显的AT编码基因。我们在此报告，通过在大肠杆菌细胞中重建 FK228 生物合成途径，两个必需基因 fabD1 和 fabD2 均编码脂肪酸合酶复合物的推定丙二酰辅酶 A (CoA) 酰基转移酶成分，被明确鉴定为参与 FK228 生物合成。任一基因产物似乎都足以补充 DepBC 上用于聚酮化合物链延长的无 AT PKS 模块。同时，编码假定的 Sfp 型磷酸泛酰胆碱基转移酶的基因 (sfp) 被鉴定为 FK228 生物合成所必需的。最有趣的是，携带可变遗传成分的工程大肠杆菌菌株在需氧和厌氧培养条件下均产生显着水平的 FK228。通过管家 AT 发现模块化 PKS 的反式互补揭示了天然产物生物合成的多样性。此外，通过工程兼性菌株厌氧生产 FK228 的证明验证了我们在新型肿瘤靶向生物制剂工程方面的努力。
Functional cross talk between fatty acid biosynthesis and secondary metabolism has been discovered in several cases in microorganisms; none of them, however, involves a modular biosynthetic enzyme. Previously, we reported a hybrid modular nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) pathway for the biosynthesis of FK228 anticancer depsipeptide in Chromobacterium violaceum strain 968. This pathway contains two PKS modules on the DepBC enzymes that lack a functional acyltransferase (AT) domain, and no apparent AT-encoding gene exists within the gene cluster or its vicinity. We report here that, through reconstitution of the FK228 biosynthetic pathway in Escherichia coli cells, two essential genes, fabD1 and fabD2, both encoding a putative malonyl coenzyme A (CoA) acyltransferase component of the fatty acid synthase complex, are positively identified to be involved in FK228 biosynthesis. Either gene product appears sufficient to complement the AT-less PKS modules on DepBC for polyketide chain elongation. Concurrently, a gene (sfp) encoding a putative Sfp-type phosphopantetheinyltransferase was identified to be necessary for FK228 biosynthesis as well. Most interestingly, engineered E. coli strains carrying variable genetic components produced significant levels of FK228 under both aerobic and anaerobic cultivation conditions. Discovery of the trans complementation of modular PKSs by housekeeping ATs reveals natural product biosynthesis diversity. Moreover, demonstration of anaerobic production of FK228 by an engineered facultative bacterial strain validates our effort toward the engineering of novel tumor-targeting bioagents.