Cloning and characterization of the tetrocarcin a gene cluster from Micromonospora chalcea NRRL 11289 reveals a highly conserved strategy for tetronate biosynthesis in spirotetronate antibiotics
Cloning and characterization of the tetrocarcin a gene cluster from Micromonospora chalcea NRRL 11289 reveals a highly conserved strategy for tetronate biosynthesis in spirotetronate antibiotics
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来自小单孢菌 NRRL 11289 的 tetrocarcin 基因簇的克隆和表征揭示了螺虫酯抗生素中特酮酸生物合成的高度保守策略
DOI：
10.1128/jb.00533-08
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发表时间：
2008-09-01
影响因子：
3.2
通讯作者：
Liu, Wen
中科院分区：
文献类型：
--
作者：
Fang, Jie;Zhang, Yiping;Liu, Wen
Tetrocarcin A（TCA）是由黄色小单孢菌（Micromonospora chalcea）NRRL 11289产生的一种螺环内酯类抗生素，具有强效的抗肿瘤活性和多种作用方式。在这项研究中，TCA的生物合成基因簇被克隆和定位到一个108 kb的连续DNA区域。计算机序列分析揭示了构成该簇的36个推定基因（包括11个用于不寻常的糖生物合成的基因，13个用于糖苷配基形成的基因和4个用于糖基化的基因），并使我们能够提出TCA的生物合成途径。D-四硝基葡萄糖、L-amicetose和L-洋地黄毒糖的形成可能开始于D-葡萄糖-1-磷酸，共享早期酶促步骤，并通过特定酶的竞争作用分支到不同的途径。四氢萘的生物合成涉及将3-C单元与聚酮化合物中间体结合以形成特征性螺四氢萘部分和反式萘烷系统。用五种脱氧糖（一种是脱氧亚硝基糖）进一步取代四氢呋喃可能是由于四种糖基转移酶的活性。在体外表征的第一个酶促步骤，利用1，3-二磷酸甘油酸作为底物和在体内交叉互补的双功能融合基因tcaD 3（与chlD 3和chlD 4的功能），以德尔塔chlD 3和德尔塔chlD 4在氯丝菌素生物合成支持螺四乙撑酯家族的高度保守的四乙撑酯的生物合成策略。编码聚酮酶的大DNA片段的缺失导致非TCA产生菌株，为鉴定新的类似物提供了明确的背景。这些研究结果提供了深入了解螺替膦酸生物合成，并证明组合生物合成方法可以应用于TCA生物合成机制，以产生结构多样性。
Tetrocarcin A (TCA), produced by Micromonospora chalcea NRRL 11289, is a spirotetronate antibiotic with potent antitumor activity and versatile modes of action. In this study, the biosynthetic gene cluster of TCA was cloned and localized to a 108-kb contiguous DNA region. In silico sequence analysis revealed 36 putative genes that constitute this cluster (including 11 for unusual sugar biosynthesis, 13 for aglycone formation, and 4 for glycosylations) and allowed us to propose the biosynthetic pathway of TCA. The formation of D-tetronitrose, L-amicetose, and L-digitoxose may begin with D-glucose-1-phosphate, share early enzymatic steps, and branch into different pathways by competitive actions of specific enzymes. Tetronolide biosynthesis involves the incorporation of a 3-C unit with a polyketide intermediate to form the characteristic spirotetronate moiety and trans-decalin system. Further substitution of tetronolide with five deoxysugars (one being a deoxynitrosugar) was likely due to the activities of four glycosyltransferases. In vitro characterization of the first enzymatic step by utilization of 1,3-biphosphoglycerate as the substrate and in vivo cross-complementation of the bifunctional fused gene tcaD3 (with the functions of chlD3 and chlD4) to Delta chlD3 and Delta chlD4 in chlorothricin biosynthesis supported the highly conserved tetronate biosynthetic strategy in the spirotetronate family. Deletion of a large DNA fragment encoding polyketide synthases resulted in a non-TCA-producing strain, providing a clear background for the identification of novel analogs. These findings provide insights into spirotetronate biosynthesis and demonstrate that combinatorial-biosynthesis methods can be applied to the TCA biosynthetic machinery to generate structural diversity.