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

• Applicant: Dr. Hajo Kries
• Subject Area: Biological and Biomimetic Chemistry
• Term: from 2020 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 441781663

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.

• DFG Programme: Research Grants