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

微生物，特别是放线菌产生的天然产物（NPs）是药物和候选药物的宝贵来源。挖掘细菌基因组揭示了NPs的大量沉默生物合成基因簇（BGCs）的存在。在合适的宿主中激活这些BGCs是一种很有希望的策略，可以获得这些未知的NPs，并满足对新药，特别是新型抗生素的需求，以应对耐药性危机。环二肽（CDP）衍生的NPs不仅因其有趣的生物学和药理学活性，而且由于其形成和修饰酶而受到越来越多的关注。在细菌中，CDPs主要由环二肽合成酶（cyclodipeptide synthases, CDPs）合成，其残基为200-300个氨基酸。cdps在遗传上与多种剪裁酶相关，包括细胞色素P450酶（P450）。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酶，作为新NPs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。申请人在过去两年内已成功确定了9个此类bgc。可以预期，更多新颖的NPs和有趣的p450是由未知的cdps相关的bgc编码的。本课题的主要目标是寻找隐藏在cdps-和p450相关bgc中的新代谢物，并鉴定新的细胞色素p450酶，这些酶可以催化cdps及其衍生物的修饰。因此，我们将研究来自系统发育树不同分支的至少含有一个CDPS和一个P450基因的15个候选bgc，这些bgc是由291个细菌P450基因参与获得的。为了克服这些基因在天然宿主中低表达或不表达以及菌株遗传操作困难的缺点，我们计划在遗传建立的彩色链霉菌中表达真实或人工BGCs。新积累的NPs将通过光谱方法分离和鉴定。随后在本地生产者中检测鉴定的代谢物可以指示bgc的表达水平。将这些产品喂给本地生产者并监测其代谢将为所识别的bgc的完整性提供证据。获得的新产品要进行各种生物试验。bgc中不同基因组合的表达和代谢物的鉴定将有助于阐明生物合成途径。p450作为关键的生物合成酶在大肠杆菌或大肠杆菌中会大量产生。在体外重建它们的活性将为这些有趣的酶的催化活性提供重要的见解。

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.