微生物次生代谢产物在控制物种相互作用和促进其防御策略方面发挥着至关重要的生态作用。它们独特的结构和强大的生物活性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与众所周知的放线菌一样，构成了一个巨大的次生代谢产物库。然而，由于大多数次生代谢物不是在实验室环境中产生的，因此迄今为止表征的粘细菌天然产物仅代表冰山一角。通过将专用的 Myxococcus xanthus DZ2 底盘菌株的工程与两步生长培养基方案相结合，我们提供了一种称为资源整合和最大化-生物分子过量生产和最佳筛选治疗的两步方案 (2PRIM-BOOST) 的新方法，用于从粘细菌中产生非核糖体肽合成酶（NRPS）/聚酮化合物合成酶（PKS）次级代谢产物。我们进一步表明，2PRIM-BOOST 策略将有助于筛选次级代谢物的医学意义的生物活性。作为概念证明，使用组成型强启动子，可以有效地生产来自 M. xanthus DZ2 的 myxoprincomide，并且使用 2PRIM-BOOST 方法增强了其生物合成，从而可以鉴定 myxoprincomide 的新特征。这种策略应该允许有机会产生和发现新的 NRPS、PKS 和混合 NRPS/PKS 混合天然代谢物，这些代谢物目前被认为是神秘的，并且在粘细菌中最具代表性。重要性微生物次生代谢物在物种相互作用中很重要，也是一种多产的代谢物。药物来源。粘细菌是普遍存在的土壤细菌，构成了巨大的次生代谢产物库。然而，由于大多数这些分子不是在实验室环境中产生的，因此可以估计迄今为止只有十分之一的分子被表征。在这里，我们开发了一种称为资源整合和最大化-生物分子过量生产和最佳筛选治疗的两步协议（2PRIM-BOOST）的新策略，该策略将专用的黄色粘球菌底盘菌株的工程与生长培养基优化结合起来。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，成功并显着增加了 myxoprincomide（一种特征性低产量次级代谢物）的产量。 2PRIM-BOOST 丰富了用于生产以前神秘的代谢物的工具箱，揭示了它们的生态作用，并提供了具有医学意义的新分子。

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 Myxococcus xanthus 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 M. xanthus 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 Myxococcus xanthus 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.