Sourice Mathieu, Simmler Charlotte, Maresca Marc, Py Béatrice, Aubert Corinne
Laboratoire de Chimie Bactérienne, UMR7283, Centre National de la Recherche Scientifique, Aix-Marseille Université, IM2B, IMM, Marseille, France.
Institut Méditerranéen de Biodiversité et d'Ecologie Marine et Continentale, UMR7263, Centre National de la Recherche Scientifique, Aix-Marseille Université, Marseille, France.
Microbiol Spectr. 2024 Oct 21;12(12):e0174024. doi: 10.1128/spectrum.01740-24.
Microbial secondary metabolites play crucial ecological roles in governing species interactions and contributing to their defense strategies. Their unique structures and potent bioactivities have been key in discovering antibiotics and anticancer drugs. Genome sequencing has undoubtedly revealed that myxobacteria constitute a huge reservoir of secondary metabolites as the well-known producers, actinomycetes. However, because most secondary metabolites are not produced in the laboratory context, the natural products from myxobacteria characterized to date represent only the tip of the iceberg. By combining the engineering of a dedicated DZ2 chassis strain with a two-step growth medium protocol, we provide a new approach called two-step Protocol for Resource Integration and Maximization-Biomolecules Overproduction and Optimal Screening Therapeutics (2PRIM-BOOST) for the production of non-ribosomal peptides synthetases (NRPS)/polyketides synthases (PKS) secondary metabolites from myxobacteria. We further show that the 2PRIM-BOOST strategy will facilitate the screening of secondary metabolites for biological activities of medical interest. As proof of concept, using a constitutive strong promoter, the myxoprincomide from DZ2 has been efficiently produced and its biosynthesis has been enhanced using the 2PRIM-BOOST approach, allowing the identification of new features of myxoprincomide. This strategy should allow the chances to produce and discover new NRPS, PKS, and mixed NRPS/PKS hybrid natural metabolites that are currently considered as cryptic and are the most represented in myxobacteria.IMPORTANCEMicrobial secondary metabolites are important in species interactions and are also a prolific source of drugs. Myxobacteria are ubiquitous soil-dwelling bacteria constituting a huge reservoir of secondary metabolites. However, because most of these molecules are not produced in the laboratory context, one can estimate that only one-tenth have been characterized to date. Here, we developed a new strategy called two-step Protocol for Resource Integration and Maximization-Biomolecules Overproduction and Optimal Screening Therapeutics (2PRIM-BOOST) that combines the engineering of a dedicated chassis strain together with growth medium optimization. By combining these strategies with the insertion of a constitutive promoter upstream the biosynthetic gene cluster (BGC), the production of myxoprincomide, a characterized low-produced secondary metabolite, was successfully and significantly increased. The 2PRIM-BOOST enriches the toolbox used to produce previously cryptic metabolites, unveil their ecological role, and provide new molecules of medical interest.
微生物次级代谢产物在调控物种相互作用和形成防御策略方面发挥着关键的生态作用。它们独特的结构和强大的生物活性一直是发现抗生素和抗癌药物的关键。基因组测序无疑揭示了粘细菌如同著名的次级代谢产物生产者放线菌一样，是次级代谢产物的巨大宝库。然而，由于大多数次级代谢产物无法在实验室环境中产生，迄今为止所鉴定出的来自粘细菌的天然产物仅仅是冰山一角。通过将专用DZ2底盘菌株的工程改造与两步生长培养基方案相结合，我们提供了一种名为“两步资源整合与最大化协议——生物分子过量生产与最佳筛选疗法”（2PRIM-BOOST）的新方法，用于从粘细菌中生产非核糖体肽合成酶（NRPS）/聚酮合酶（PKS）次级代谢产物。我们进一步表明，2PRIM-BOOST策略将有助于筛选具有医学意义生物活性的次级代谢产物。作为概念验证，利用组成型强启动子，已高效生产出DZ2菌株的粘菌素，并使用2PRIM-BOOST方法增强了其生物合成，从而得以鉴定出粘菌素的新特性。该策略应能增加生产和发现新型NRPS、PKS以及混合NRPS/PKS杂合天然代谢产物的机会，这些产物目前被视为隐秘产物，且在粘细菌中最为常见。
重要性
微生物次级代谢产物在物种相互作用中很重要，也是丰富的药物来源。粘细菌是普遍存在于土壤中的细菌，是次级代谢产物的巨大宝库。然而，由于这些分子大多无法在实验室环境中产生，可以估计迄今为止只有十分之一被鉴定出来。在此，我们开发了一种名为“两步资源整合与最大化协议——生物分子过量生产与最佳筛选疗法”（2PRIM-BOOST）的新策略，该策略将专用底盘菌株的工程改造与生长培养基优化相结合。通过将这些策略与在生物合成基因簇（BGC）上游插入组成型启动子相结合，成功且显著地提高了已鉴定的低产次级代谢产物粘菌素的产量。2PRIM-BOOST丰富了用于生产先前隐秘代谢产物的工具库，揭示了它们的生态作用，并提供了具有医学意义的新分子。
