Colistin, also known as polymyxin E, is a lipopeptide antibiotic used to treat infections caused by multidrug-resistant gram-negative bacteria. It is considered a “last-line antibiotic”, but its clinical development is hindered by low titer and impurities resulting from the presence of diverse homologs in microbial fermentation. To ensure consistent pharmaceutical activity and kinetics, it is crucial to have high-purity colistin active pharmaceutical ingredient (API) in the pharmaceutical industry. This study focused on the metabolic engineering of a natural colistin producer strain to produce colistin with a high titer and purity. Guided by genome mining, we identified Paenibacillus polymyxa ATCC 842 as a natural colistin producer capable of generating a high proportion of colistin A. By systematically inactivating seven non-essential biosynthetic gene clusters (BGCs) of peptide metabolites that might compete precursors with colistin or inhibit colistin production, we created an engineered strain, P14, which exhibited an 82% increase in colistin titer and effectively eliminated metabolite impurities such as tridecaptin, paenibacillin, and paenilan. Additionally, we engineered the L-2,4-diaminobutyric acid (L-2,4-DABA) pathway to further enhance colistin production, resulting in the engineered strain P19, which boosted a remarkable colistin titer of 649.3 mg/L – a 269% improvement compared to the original strain. By concurrently feeding L-isoleucine and L-leucine, we successfully produced high-purity colistin A, constituting 88% of the total colistin products. This study highlights the potential of metabolic engineering in improving the titer and purity of lipopeptide antibiotics in the non-model strain, making them more suitable for clinical use. These findings indicate that efficiently producing colistin API in high purity directly from fermentation can now be achieved in a straightforward manner.

中文翻译：

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多粘菌 Paenibacillus 中“最后一线抗生素”粘菌素的代谢工程


粘菌素，也称为多粘菌素 E，是一种脂肽抗生素，用于治疗由多重耐药革兰氏阴性菌引起的感染。它被认为是“最后一线抗生素”，但其临床开发受到低滴度和微生物发酵中存在多种同源物导致的杂质的阻碍。为了确保一致的药物活性和动力学，制药行业拥有高纯度粘菌素活性药物成分 （API） 至关重要。本研究的重点是天然粘菌素生产菌株的代谢工程，以产生具有高滴度和纯度的粘菌素。在基因组挖掘的指导下，我们确定多粘菌 Paenibacillus ATCC 842 是一种天然粘菌素生产者，能够产生高比例的粘菌素 A。通过系统灭活可能与粘菌素竞争前体或抑制粘菌素产生的肽代谢物的七个非必需生物合成基因簇 （BGC），我们创造了一种工程菌株 P14，其粘菌素滴度增加了 82%，并有效消除了代谢物杂质，如十三卡普汀、帕尼西林和帕尼兰。此外，我们设计了 L-2,4-二氨基丁酸 （L-2,4-DABA） 途径以进一步增强粘菌素的产生，从而产生了工程菌株 P19，其粘菌素滴度显著提高了 649.3 mg/L，与原始菌株相比提高了 269%。通过同时补料 L-异亮氨酸和 L-亮氨酸，我们成功生产了高纯度粘菌素 A，占粘菌素总产物的 88%。本研究强调了代谢工程在提高非模型菌株中脂肽抗生素的滴度和纯度方面的潜力，使其更适合临床使用。 这些发现表明，现在可以以直接的方式直接从发酵中高效生产高纯度粘菌素 API。