Novel metabolites hidden in the cyclodipeptide-related biosynthetic gene clusters and intriguing reactions catalyzed by cytochrome P450 enzymes

隐藏在环二肽相关生物合成基因簇中的新代谢物以及细胞色素P450酶催化的有趣反应

• 批准号: 444837129
• 负责人: Professor Dr. Shu-Ming Li
• 金额: --
• 依托单位: Institut für Pharmazeutische Biologie und Biotechnologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/444837129?language=en
• 关键词: Novel; metabolites; hidden; cyclodipeptide; related

Natural products (NPs) produced by microorganisms, especially by actinobacteria, are valuable sources of drugs and candidates. Mining the bacterial genomes revealed the presence of a large number of silent biosynthetic gene clusters (BGCs) for NPs. Activation of such BGCs in an appropriate host is a promising strategy to access these unknown NPs and to meet the need of new drugs, especially of novel antibiotics to tackle the resistance crisis. Cyclodipeptide (CDP)-derived NPs have gained increasing attention not only due to their interesting biological and pharmacological activities but also because of their forming and modification enzymes. In bacteria, CDPs are biosynthesized mainly by cyclodipeptide synthases (CDPSs) with 200–300 amino acid residues. The CDPSs are genetically associated with a variety of tailoring enzymes including cytochrome P450 enzymes (P450s). Interestingly, the small cdps-associated BGCs code often for complex and fascinating chemical structures. The P450s from these BGCs, as responsible enzymes for the novel NPs, catalyze unusual reactions such as aryl-aryl coupling, N-oxide formation and coupling of indole with nucleobases. Nine such BGCs have been successfully identified by the applicant in the last two years. It can be expected that more novel NPs and intriguing P450s are encoded by unknown cdps-associated BGCs.The main goal of this proposal is finding novel metabolites hidden in the cdps- and P450-associated BGCs and identification of new cytochrome p450 enzymes, which catalyze fascinating modifications of CDPs and derivatives thereof. Therefore, 15 candidate BGCs containing at least one CDPS and one P450 gene from different clades of a phylogenetical tree will be investigated, which was obtained by involvement of 291 bacterial P450s. To overcome the disadvantages of low or non-expression of the genes in the native hosts and often difficult genetic manipulation of the strains, we plan to express the genuine or artificial BGCs in the genetically established Streptomyces coelicolor. The newly accumulated NPs will be isolated and identified by spectroscopic methods. Detection of the identified metabolites in the native producer afterward can give indications for the expression level of the BGCs. Feeding these products into the native producer and monitoring their metabolism will provide evidence for the integrity of the identified BGCs. The obtained new products are subjected to diverse biological tests. Expression of different combinations of the genes from the BGCs and identification of the metabolites will help to elucidate the biosynthetic pathways. P450s as the key biosynthetic enzymes will be overproduced in E. coli or S. coelicolor. In vitro reconstruction of their activities will provide important insights into the catalytic activities of these intriguing enzymes.

由微生物特别是放线菌产生的天然产物（NP）是药物和候选药物的宝贵来源。对细菌基因组的挖掘揭示了大量用于NP的沉默生物合成基因簇（BGC）的存在。在适当的宿主中激活这样的BGC是一种有希望的策略，以获得这些未知的NP并满足对新药的需求，特别是对新型抗生素的需求，以解决耐药性危机。环二肽衍生的纳米粒不仅具有重要的生物学和药理学活性，而且具有形成和修饰酶的功能，因此受到越来越多的关注。在细菌中，CDP主要由具有200-300个氨基酸残基的环二肽脱氢酶（CDPS）生物合成。CDPS与包括细胞色素P450酶（P450）在内的多种剪裁酶遗传相关。有趣的是，与cdps相关的小bgc通常编码复杂而迷人的化学结构。来自这些BGC的P450作为新型NP的负责酶，催化不寻常的反应，如芳基-芳基偶联、N-氧化物形成和吲哚与核碱基的偶联。在过去两年，申请人已成功找到九间这类英国政府总部。本研究的主要目标是寻找隐藏在cdps和P450相关的BGC中的新的代谢物，并鉴定新的细胞色素p450酶，这些酶催化CDPs及其衍生物的有趣修饰。因此，将研究15个候选BGC，这些候选BGC含有来自不同进化枝的至少一个CDPS和一个P450基因，其通过涉及291个细菌P450而获得。为了克服这些基因在天然宿主中表达量低或不表达的缺点以及菌株的遗传操作困难，我们计划在遗传上建立的天蓝色链霉菌中表达真正的或人工的BGC。新积累的NP将通过光谱方法分离和鉴定。随后在天然生产者中检测鉴定的代谢物可以给出BGC表达水平的指示。将这些产品喂入本地生产者并监测其代谢将为所鉴定的BGC的完整性提供证据。获得的新产品经过各种生物学试验。从BGC的基因的不同组合的表达和代谢产物的鉴定将有助于阐明生物合成途径。P450作为生物合成的关键酶，在大肠杆菌中过量产生。coli或S.天蓝色。在体外重建他们的活动将提供重要的见解，这些有趣的酶的催化活性。