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

通过放线菌宏基因组和靶向代谢组分析建立烯啶生物合成工具箱

• 批准号: 276972917
• 负责人: Dr. Ivana Crnovcic
• 金额: --
• 依托单位: Department of Chemistry
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/276972917?language=en
• 关键词: Approach; enedyine; biosynthesis; tool; box

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.

Enediynes是由土壤和海洋微生物产生的一类有效的抗肿瘤药物。在结构上，它们的特征是由两个乙炔基团组成的核心单元，这些基团共轭到9元或10元环中的双键或初始双键。烯二炔类化合物基于其烯二炔核的存在而具有抗癌和抗菌活性，而不同的烯二炔类化合物核心周围的外围化学仅在该活性的微调中起作用。Enediynes在DNA链间DNA交联物(ICL)中产生，并可导致位点特异性的单链或双链断裂(DSB)。对已知的烯二炔生物合成基因簇的比较表明，9元和10元烯二炔都有一个保守的五基因盒，即烯二炔PKS盒，它编码了烯二炔核心结构的十五碳七烯骨架的生物合成。十五碳十七烯中心核心的保守性受到外围部分的干扰，这导致了各种烯二炔的高度结构多样性。由于烯二炔类化合物作为纯化合物具有极强的细胞毒活性，它们作为临床药物的用途有限。然而，当它们被制成各种基于聚合物的递送系统或作为抗癌抗体-药物结合物(ADCs)时，已经显示出巨大的临床成功，并在未来的抗癌治疗中具有非常大的发展前景。因此，开发具有更好性能的新型烯二炔化合物的生物合成具有重要意义。沈教授的团队对他们收集的3200株放线菌进行了基因组调查，并确定了89株含有烯二炔PKS基因盒的菌株。具体地说，我们将从该菌株集合中选择两株命名为CB02115和CB100675的菌株作为模型，以研究其生物合成中的特定类群或功能基团等新的结构特征。CB02115编码的基因簇与球孢链霉菌的C-1027基因簇相似，而CB100675编码的基因簇与棘球小单孢菌的钙霉素(CAL)基因簇相似。为了研究菌株CB02115和菌株CB100675中烯二炔基因簇编码的烯二炔的结构和生物合成特征，我们将确定新的烯二炔的结构，并分析在烯二炔生物合成中具有明显新生物合成功能的基因。此外，鉴于两个新的烯二炔基因簇与已知簇的相似性，我们将在遗传背景下表达基因构建物，积极合成常规的烯二炔类化合物。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该基因并在体外用合适的模型底物测试酶来鉴定其基因产物。

项目成果

Strain Prioritization and Genome Mining for Enediyne Natural Products.

• DOI: 10.1128/mbio.02104-16
• 发表时间: 2016-12-20
• 期刊: mBio
• 影响因子: 6.4
• 作者: Yan X;Ge H;Huang T;Hindra;Yang D;Teng Q;Crnovčić I;Li X;Rudolf JD;Lohman JR;Gansemans Y;Zhu X;Huang Y;Zhao LX;Jiang Y;Van Nieuwerburgh F;Rader C;Duan Y;Shen B
• 通讯作者: Shen B

Engineered production of cancer targeting peptide (CTP)-containing C-1027 in Streptomyces globisporus and biological evaluation.

靶向肽（CTP）的工程生产 - 全球链霉菌和生物学评估中含有C-1027。

• DOI: 10.1016/j.bmc.2016.04.017
• 发表时间: 2016-09-01
• 期刊: Bioorganic & medicinal chemistry
• 影响因子: 3.5
• 作者: Li W;Li X;Huang T;Teng Q;Crnovcic I;Rader C;Shen B
• 通讯作者: Shen B

Discovery of Alternative Producers of the Enediyne Antitumor Antibiotic C-1027 with High Titers.

• DOI: 10.1021/acs.jnatprod.7b01013
• 发表时间: 2018-03-23
• 期刊: Journal of natural products
• 影响因子: 5.1
• 作者: Yan X;Hindra;Ge H;Yang D;Huang T;Crnovcic I;Chang CY;Fang SM;Annaval T;Zhu X;Huang Y;Zhao LX;Jiang Y;Duan Y;Shen B
• 通讯作者: Shen B