Polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS) comprise biosynthetic pathways that provide access to diverse, often bioactive natural products. Metabolic engineering can improve production metrics to support characterization and drug-development studies, but often native hosts are difficult to genetically manipulate and/or culture. For this reason, heterologous expression is a common strategy for natural product discovery and characterization. Many bacteria have been developed to express heterologous biosynthetic gene clusters (BGCs) for producing polyketides and nonribosomal peptides. In this article, we describe tools for using Pseudomonas putida, a Gram-negative soil bacterium, as a heterologous host for producing natural products. Pseudomonads are known to produce many natural products, but P. putida production titers have been inconsistent in the literature and often low compared to other hosts. In recent years, synthetic biology tools for engineering P. putida have greatly improved, but their application towards production of natural products is limited. To demonstrate the potential of P. putida as a heterologous host, we introduced BGCs encoding the synthesis of prodigiosin and glidobactin A, two bioactive natural products synthesized from a combination of PKS and NRPS enzymology. Engineered strains exhibited robust production of both compounds after a single chromosomal integration of the corresponding BGC. Next, we took advantage of a set of genome-editing tools to increase titers by modifying transcription and translation of the BGCs and increasing the availability of auxiliary proteins required for PKS and NRPS activity. Lastly, we discovered genetic modifications to P. putida that affect natural product synthesis, including a strategy for removing a carbon sink that improves product titers. These efforts resulted in production strains capable of producing 1.1 g/L prodigiosin and 470 mg/L glidobactin A.

聚酮化合物合成酶 (PKS) 和非核糖体肽合成酶 (NRPS) 包含生物合成途径，可提供获得多种通常具有生物活性的天然产物的途径。代谢工程可以改善生产指标以支持表征和药物开发研究，但通常天然宿主难以进行基因操作和/或培养。因此，异源表达是天然产物发现和表征的常用策略。已经开发了许多细菌来表达异源生物合成基因簇 (BGC) 以产生聚酮化合物和非核糖体肽。在本文中，我们描述了使用恶臭假单胞菌的工具，一种革兰氏阴性土壤细菌，作为生产天然产物的异源宿主。已知假单胞菌会产生许多天然产物，但文献中恶臭假单胞菌的生产滴度不一致，并且与其他宿主相比通常较低。近年来，用于工程化恶臭假单胞菌的合成生物学工具有了很大的改进，但它们在天然产物生产中的应用有限。展示恶臭假单胞菌的潜力作为异源宿主，我们引入了编码灵菌红素和格列杆菌素 A 合成的 BGC，这两种生物活性天然产物由 PKS 和 NRPS 酶学组合合成。工程菌株在相应 BGC 的单一染色体整合后表现出两种化合物的强劲生产。接下来，我们利用一组基因组编辑工具通过修改 BGC 的转录和翻译以及增加 PKS 和 NRPS 活性所需的辅助蛋白的可用性来增加滴度。最后，我们发现了恶臭假单胞菌的基因修饰影响天然产物合成的，包括去除碳汇以提高产品效价的策略。这些努力产生了能够生产 1.1 g/L 灵菌红素和 470 mg/L 格利多菌素 A 的生产菌株。