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

非核糖体肽合成酶(NRPS)是一类参与合成多种生物活性天然产物的酶，如环孢素、万古霉素和表面活性物质等。通过NRPS工程，这些化合物可能会在结构上多样化，并针对某些应用而量身定做。为此，我们将拓宽腺化(A-)结构域的底物光谱，该结构域在NRPS中主要负责底物选择。这种方法是基于这样一种假设，即混杂酶在自然界和实验室中是多功能的进化中间体。在定向进化实验中，我们将从表面素生物合成中突变模块SrfA-C的A-结构域，同时通过异羟甲酸酯图谱(HAMA)监测数百个突变体的完整底物光谱。HAMA通过质谱学来解开当淬灭剂羟胺加入A-结构域反应时形成的异羟甲酸酯产物。使用HAMA，可以并行测试数十种底物，这反映了细胞内部的情况，并在一次实验中产生了A结构域的全面特异谱。SrfA-C定向进化为高度混杂的“sfA-Chub”--一个达到各种特异性的多功能枢纽--将在HAMA指导下进行几轮连续的突变。基于在此过程中构建的突变文库的HAMA图谱，我们将量化单个A-结构域残基对特异性的影响，以指导未来的定向进化实验。进化的srfA-Chub将提供一个混杂进化中间体的人工模型，其特征将有助于我们理解自然的NRPS进化。SfA-Chub还将提供一个蓝图，以加强相关A域中的混杂。为了测试表面素变异体的形成，将在天然表面素生产者中用srfA-Chub取代srfA-C基因。总之，这些实验将增进我们对NRPS工程和进化的理解，从而为未来定制救命多肽药物的生物合成奠定基础。

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.