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。对细菌基因组的挖掘发现，存在大量针对NPs的沉默的生物合成基因簇(BGC)。在合适的宿主中激活这些BGC是一种很有前途的策略，可以获得这些未知的NPs，并满足新药的需求，特别是解决耐药性危机的新型抗生素。环二肽(CDP)衍生的纳米粒子不仅因为其有趣的生物学和药理活性，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，CDP主要由含有200-300个氨基酸残基的环二肽合成酶(CDPS)合成。CDPS在基因上与多种剪裁酶有关，包括细胞色素P450酶(P450)。有趣的是，小的CDP相关的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NPs的负责酶，能够催化不寻常的反应，如芳基-芳基偶联、N-氧化物的形成以及吲哚与碱基的偶联。在过去两年，申请人已成功辨认出九个这类英国属土公民。可以预期，更多新的NPs和有趣的P450是由未知的CDPs相关的BGC编码的。本提案的主要目标是寻找隐藏在CDPs和P450相关的BGC中的新代谢物，并鉴定新的细胞色素P450酶，这些酶催化CDPs及其衍生物的迷人修饰。因此，从一个系统发育树的不同分支中获得的15个候选BGC将被调查，这些BGC至少包含一个CDP和一个P450基因，这些基因是由291个细菌P450参与获得的。为了克服基因在天然宿主中低表达或不表达的缺点，以及菌株通常难以进行遗传操作的缺点，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造的BGC。新积累的NPs将通过光谱方法进行分离和鉴定。随后对本地生产者中已鉴定的代谢物的检测可以给出BGC的表达水平的指示。将这些产品输入本地生产商并监测它们的新陈代谢，将为已确定的BGC的完整性提供证据。获得的新产品经过了各种生物测试。BGC基因的不同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作为生物合成的关键酶，将在大肠杆菌或天蓝色链霉菌中大量生产。在体外重建它们的活性将为了解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.