Stepwise genetic engineering of Pseudomonas putida enables robust heterologous production of prodigiosin and glidobactin A.
Stepwise genetic engineering of Pseudomonas putida enables robust heterologous production of prodigiosin and glidobactin A.
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假单胞菌的逐步遗传工程使Protigiosin和Glidobactin A的强大异源产生A。
DOI：
10.1016/j.ymben.2021.06.004
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发表时间：
2021-09
影响因子：
8.4
通讯作者：
Pfleger BF
中科院分区：
文献类型：
--
作者：
Cook TB;Jacobson TB;Venkataraman MV;Hofstetter H;Amador-Noguez D;Thomas MG;Pfleger BF
聚酮合成酶（PKS）和非核糖体肽合成酶（NRPS）构成了生物合成途径，提供了获得多种生物活性天然产物的途径。代谢工程可以改善生产指标，以支持表征和药物开发研究，但通常原生宿主难以基因操纵和/或培养。因此，异源表达是天然产物发现和表征的常用策略。许多细菌已经发展到表达异源生物合成基因簇（BGCs）来生产多酮和非核糖体肽。在本文中，我们描述了利用革兰氏阴性土壤细菌恶臭假单胞菌作为异源宿主生产天然产物的工具。众所周知，假单胞菌可以产生许多天然产物，但在文献中，恶臭假单胞菌的生产滴度一直不一致，与其他宿主相比，它的生产滴度往往较低。近年来，工程化恶臭杆菌的合成生物学工具有了很大的改进，但在天然产物生产中的应用有限。为了证明恶臭p.p . putida作为异源寄主的潜力，我们引入了编码合成prodigiosin和glidobactin a的bgc，这两种生物活性天然产物是由PKS和NRPS酶学结合合成的。工程菌株在单个染色体整合相应的BGC后显示出这两种化合物的强大生产。接下来，我们利用一套基因组编辑工具，通过修改bgc的转录和翻译，增加PKS和NRPS活性所需的辅助蛋白的可用性，来提高滴度。最后，我们发现了影响天然产物合成的恶臭假单胞菌的遗传修饰，包括去除碳汇以提高产物滴度的策略。这些努力使生产菌株能够产生1.1 g/L的芥子菌素和470 mg/L的glidobactin A。
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.
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影响因子：
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作者：
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通讯作者：
Rabinowitz, Joshua D
影响因子：
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