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Combinatorial metabolic engineering of Bacillus subtilis for de novo production of polymyxin B
Metabolic Engineering ( IF 6.8 ) Pub Date : 2024-04-04 , DOI: 10.1016/j.ymben.2024.04.001
Hui-Zhong Sun 1 , Qing Li 1 , Wei Shang 1 , Bin Qiao 1 , Qiu-Man Xu 2 , Jing-Sheng Cheng 1
Affiliation  

Polymyxin is a lipopeptide antibiotic that is effective against multidrug-resistant Gram-negative bacteria. However, its clinical development is limited due to low titer and the presence of homologs. To address this, the polymyxin gene cluster was integrated into Bacillus subtilis, and sfp from Paenibacillus polymyxa was expressed heterologously, enabling recombinant B. subtilis to synthesize polymyxin B. Regulating NRPS domain inhibited formation of polymyxin B2 and B3. The production of polymyxin B increased to 329.7 mg/L by replacing the native promoters of pmxA, pmxB, and pmxE with PfusA, C2up, and PfusA, respectively. Further enhancement in this production, up to 616.1 mg/L, was achieved by improving the synthesis ability of 6-methyloctanoic acid compared to the original strain expressing polymyxin heterologously. Additionally, incorporating an anikasin-derived domain into the hybrid nonribosomal peptide synthase of polymyxin increased the B1 ratio in polymyxin B from 57.5% to 62.2%. Through optimization of peptone supply in the fermentation medium and fermentation in a 5.0-L bioreactor, the final polymyxin B titer reached 962.1 mg/L, with a yield of 19.24 mg/g maltodextrin and a productivity of 10.02 mg/(L·h). This study demonstrates a successful approach for enhancing polymyxin B production and increasing the B1 ratio through combinatorial metabolic engineering.

中文翻译:


枯草芽孢杆菌的组合代谢工程用于从头生产多粘菌素 B



多粘菌素是一种脂肽抗生素,对多重耐药革兰氏阴性菌有效。然而,由于低滴度和同源物的存在,其临床开发受到限制。为了解决这个问题,将多粘菌素基因簇整合到枯草芽孢杆菌中,并且来自 Paenibacillus polymyxa 的 sfp 异源表达,使重组枯草芽孢杆菌能够合成多粘菌素 B。通过分别用 PfusA、C2up 和 PfusA 取代 pmxA 、 pmxB 和 pmxE 的天然启动子,多粘菌素 B 的产量增加到 329.7 mg/L。与异源表达多粘菌素的原始菌株相比,通过提高 6-甲基辛酸的合成能力,实现了该产量的进一步增强,高达 616.1 mg/L。此外,将异香蛋白衍生结构域掺入多粘菌素的杂化非核糖体肽合酶中,可将多粘菌素 B 中的 B1 比率从 57.5% 提高到 62.2%。通过优化发酵培养基中的蛋白胨供应,并在 5.0 L 生物反应器中发酵,多粘菌素 B 最终滴度达到 962.1 mg/L,麦芽糖糊精产量为 19.24 mg/g,产能为 10.02 mg/(L·h)。本研究展示了一种通过组合代谢工程增强多粘菌素 B 产生和增加 B1 比率的成功方法。
更新日期:2024-04-04
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