Approach to a enedyine biosynthesis tool box through metagenomic and targeted metabolomic analyses of actinomycetes

烯二炔是一类由土壤和海洋微生物产生的强效抗肿瘤药物。在结构上，它们的特点是在一个9元或10元的环内，由两个乙基组成的核心单元与一个双键或初双键共轭。enediynes具有抗癌和抗生素活性，这是基于它们的enediyne核心的存在，而在不同enediynes核心周围发现的外周化学物质仅在这种活性的微调中起作用。Enediynes在DNA链间产生DNA交联（ICLs），并可导致位点特异性单链或双链断裂（DSBs）。对已知的烯二炔生物合成基因簇的比较表明，9元和10元烯二炔共享一个保守的5基因盒，即烯二炔PKS盒，编码烯二炔核心结构的pentadecaheptaene主链的生物合成。pentadecaheptaene中央核心的保存受到外围部分的干扰，这导致了各种烯的高度结构多样性。由于烯二炔作为纯化合物具有极强的细胞毒活性，因此它们作为临床药物的使用有限。然而，当它们被配制成各种基于聚合物的递送系统或作为抗癌抗体-药物偶联物（adc）时，它们已经显示出巨大的临床成功，并且在抗癌治疗的未来发展中非常有希望。因此，生物合成开发性能更好的新型烯二炔类化合物具有重要意义。沈教授的研究小组对他们收集的3200株放线菌进行了基因组调查，发现了89株含有enediyne PKS基因磁带。具体而言，我们将从该菌株收集中选择两株CB02115和CB100675作为模型来研究其生物合成中的新结构特征，例如特定的类群或功能基团。CB02115编码的基因簇与链霉菌globisporus的C-1027基因簇相似，而CB100675编码的基因簇与棘孢小单孢菌calicheamicin （CAL）基因簇相似。为了研究菌株CB02115和菌株CB100675中enediyne基因簇编码的enediyne的结构和生物合成特征，我们将确定新enediyne的结构并分析在enediyne生物合成中具有明显新生物合成功能的有前景的基因。此外，鉴于两个新的烯二炔基因簇与已知簇的相似性，我们将在积极合成传统烯二炔的遗传背景下表达基因构建。对于CB02115或CB100675中最成功的基因，我们将通过在大肠杆菌中表达基因并在体外用合适的模型底物测试酶来体外表征其基因产物。

Enediynes are a class of potent antitumor agents produced by soil and marine microorganisms. Structurally they are characterized by a core unit consisting of two acetylenic groups conjugated to a double bond or incipient double bond within a 9- or 10-membered ring. The enediynes possess anticancer and antibiotic activity based on the presence of their enediyne core whereas the peripheral chemistry found around the cores of the different enediynes plays only a role in the fine tuning of that activity. Enediynes produce in the DNA inter-strand DNA crosslinks (ICLs) and can lead to site-specific single stranded or double stranded breaks (DSBs). Comparison of the known enediyne biosynthetic gene clusters has revealed that both, 9- and 10-membered enediynes, share a conserved five-gene cassette, the enediyne PKS cassette, which encodes the biosynthesis of the pentadecaheptaene backbone of the enediyne core structure. The conservation of the central core of the pentadecaheptaene is disturbed by the peripheral parts which lead to a high structural diversity of the various enediynes. Since the enediynes have extreme cytotoxic activity as pure compounds they have only limited use as clinical drugs. However, when formulated into various polymer-based delivery systems or as anticancer antibody-drug conjugates (ADCs) they have shown great clinical success and are very promising for future developments in anticancer therapy. Therefore the biosynthetic development of new enediyne compounds with still better performance is of interest. The group of Prof. Shen has done a genome survey of 3,200 strains of their Actinomycetale collection and identified 89 strains that harbor the enediyne PKS gene cassettes. In detail, two strains, named CB02115 and CB100675 from this strain collection we will chose as models to investigate novel structural features such as e. g. specific groupings or functional groups in their biosynthesis. CB02115 encodes a gene cluster that resembles the C-1027 gene cluster from Streptomyces globisporus, while CB100675 encodes a gene cluster that shows similarity to the calicheamicin (CAL) gene cluster from Micromonospora echinospora. To address the structure and the biosynthetic features of the enediynes encoded by the enediyne gene cluster in strain CB02115 and strain CB100675 we will determine the structures of the new enediynes and analyse promising genes with apparent novel biosynthetic function in enediyne biosynthesis. Moreover, in view of the similarity of the two new enediyne gene clusters with already known clusters we will express the gene constructs in a genetic background actively synthesizing conventional enediynes. For the most successful genes in either CB02115 or CB100675 we will in vitro characterize its gene product by expressing the gene in E. coli and testing the enzyme with a suitable model substrate in vitro.