National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China.
National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China.
Bioresour Technol. 2024 Feb;394:130220. doi: 10.1016/j.biortech.2023.130220. Epub 2023 Dec 16.
Pseudomonas putida KT2440, a GRAS strain, has been used for synthesizing bulk and fine chemicals. However, the gene editing tool to metabolically engineer KT2440 showed low efficiency. In this study, a novel sacB-based system pK51mobsacB was established to improve the efficiency for marker-free gene disruption. Then the rhamnolipid synthetic pathway was introduced in KT2440 and genes of the competitive pathways were deleted to lower the metabolic burden based on pK51mobsacB. A series of endogenous and synthetic promoters were used for fine tuning rhlAB expression. The limited supply of dTDP-L-rhamnose was enhanced by heterologous rmlBDAC expression. Cell growth and rhamnolipid production were well balanced by using glucose/glycerol as mixed carbon sources. The final strain produced 3.64 g/L at shake-flask and 19.77 g/L rhamnolipid in a 5 L fermenter, the highest obtained among metabolically engineered KT2440, which implied the potential of KT2440 as a promising microbial cell factory for industrial rhamnolipid production.
恶臭假单胞菌 KT2440 是一种 GRAS 菌株，已被用于合成大宗化学品和精细化学品。然而，用于代谢工程 KT2440 的基因编辑工具显示出低效率。在本研究中，建立了一种新型的 sacB 为基础的 pK51mobsacB 系统，以提高无标记基因敲除的效率。然后，基于 pK51mobsacB 将鼠李糖脂合成途径引入 KT2440 中，并删除竞争途径的基因，以降低代谢负担。一系列内源和合成启动子用于精细调节 rhlAB 的表达。通过异源 rmlBDAC 的表达增强了 dTDP-L-鼠李糖的有限供应。通过使用葡萄糖/甘油作为混合碳源，很好地平衡了细胞生长和鼠李糖脂的生产。最终的菌株在摇瓶中生产了 3.64 g/L，在 5 L 发酵罐中生产了 19.77 g/L 的鼠李糖脂，这是代谢工程 KT2440 中获得的最高产量，这表明 KT2440 作为一种有前途的工业鼠李糖脂生产微生物细胞工厂的潜力。
