Directed Evolution of Adenylation-Domains Guided by Hydroxamate Profiling (DEHAMA)

异羟肟酸分析引导的腺苷酸化结构域定向进化 (DEHAMA)

• 批准号: 441781663
• 负责人: Dr. Hajo Kries
• 金额: --
• 依托单位: Abteilung Biomolekulare Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/441781663?language=en
• 关键词: Directed; Evolution; Adenylation; Domains; Guided

Nonribosomal peptide synthetases (NRPS) are enzymes involved in the production of multifarious bioactive natural products, such as ciclosporin, vancomycin and surfactin. Through NRPS engineering, these compounds can potentially be structurally diversified and tailored to certain applications. To this end we will broaden the substrate spectrum of the adenylation (A-) domain which is primarily responsible for substrate selection in NRPS. This approach is based on the hypothesis that promiscuous enzymes are versatile evolutionary intermediates in nature and in the lab. We will mutate the A-domain in module SrfA-C from surfactin biosynthesis in a directed evolution experiment while monitoring the full substrate spectrum of hundreds of mutants via hydroxamate profiling (HAMA). HAMA disentangles by mass spectrometry which hydroxamate products are formed when the quencher hydroxylamine is added to an A-domain reaction. With HAMA, dozens of substrates can be tested in parallel which reflects the situation inside a cell and yields a comprehensive specificity profile of an A-domain in a single experiment. Directed evolution of SrfA-C to the highly promiscuous “SrfA-Chub” - a versatile hub to reach various specificities - will be performed in several consecutive rounds of mutagenesis guided by HAMA. Based on HAMA profiles of mutational libraries constructed in the process, we will quantify the influence of individual A-domain residues on specificity to inform future directed evolution experiments. The evolved SrfA-Chub will provide an artificial model of a promiscuous evolutionary intermediate and its characterization will contribute to our understanding of natural NRPS evolution. SrfA-Chub will also provide a blueprint to enhance promiscuity in related A-domains. To test for formation of surfactin variants, the srfA-C gene will be replaced with srfA-Chub in the natural surfactin producer. Altogether, these experiments will advance our understanding of NRPS engineering and evolution, thus laying the basis for tailoring the biosynthesis of life-saving peptide drugs in the future.

非核糖体肽合成酶（NRPS）是一类参与环孢菌素、万古霉素和表面活性素等多种生物活性天然产物合成的酶。通过NRPS工程，这些化合物可以潜在地在结构上多样化并针对某些应用进行定制。为此，我们将拓宽底物谱的腺苷酸化（A-）结构域，这是主要负责底物选择NRPS。这种方法是基于这样的假设，即混杂酶是自然界和实验室中通用的进化中间体。我们将在定向进化实验中突变来自表面活性素生物合成的模块SrfA-C中的A结构域，同时通过异羟肟酸分析（HAMA）监测数百种突变体的全底物谱。HAMA通过质谱法解缠结，当将猝灭剂羟胺加入到A结构域反应中时形成异羟肟酸产物。使用HAMA，可以并行测试数十种底物，这反映了细胞内的情况，并在单个实验中产生A结构域的全面特异性谱。将SrfA-C定向进化为高度混杂的“SrfA-Chub”--一种达到各种特异性的通用枢纽--将在HAMA指导的几轮连续诱变中进行。基于该过程中构建的突变库的HAMA谱，我们将量化单个A结构域残基对特异性的影响，为未来的定向进化实验提供信息。进化的SrfA-Chub将提供一个混杂进化中间体的人工模型，其表征将有助于我们对自然NRPS进化的理解。SrfA-Chub还将提供一个蓝图，以增强相关A域的混杂性。为了测试表面活性素变体的形成，将天然表面活性素生产者中的srfA-C基因替换为srfA-Chub。总而言之，这些实验将促进我们对NRPS工程和进化的理解，从而为未来定制救生肽药物的生物合成奠定基础。