Xie Wen-Liang, Zhang Mei-Fang, Huang Zheng-Yu, Xu Man, Li Chun-Xiu, Xu Jian-He
Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
Bioresour Bioprocess. 2024 May 16;11(1):50. doi: 10.1186/s40643-024-00762-8.
Biosynthesis of paclitaxel (Taxol™) is a hot topic with extensive and durable interests for decades. However, it is severely hindered due to the very low titers of intermediates. In this study, Escherichia coli was employed to de novo synthesize a key intermediate of paclitaxel, taxadien-5α-yl-acetate (T5OAc). Plasmid-based pathway reconstruction and optimization were conducted for T5OAc production. The endogenous methylerythritol phosphate pathway was enhanced to increase the precursor supply. Three taxadien-5α-ol O-acetyltransferases were tested to obtain the best enzyme for the acetylation step. Metabolic burden was relieved to restore cell growth and promote production through optimizing the plasmid production system. In order to achieve metabolic balance, the biosynthesis pathway was regulated precisely by multivariate-modular metabolic engineering. Finally, in a 5-L bioreactor, the T5OAc titer was enhanced to reach 10.9 mg/L. This represents an approximately 272-fold increase in production compared to the original strain, marking the highest yield of T5OAc ever documented in E. coli, which is believed to be helpful for promoting the progress of paclitaxel biosynthesis.
几十年来，紫杉醇（泰素™）的生物合成一直是一个备受广泛且持久关注的热门话题。然而，由于中间体的产量极低，其生物合成受到严重阻碍。在本研究中，利用大肠杆菌从头合成紫杉醇的关键中间体5α-乙酰氧基紫杉二烯（T5OAc）。针对T5OAc的生产进行了基于质粒的途径重建和优化。增强了内源性甲基赤藓糖醇磷酸途径以增加前体供应。测试了三种5α-羟基紫杉二烯O-乙酰基转移酶，以获得用于乙酰化步骤的最佳酶。通过优化质粒生产系统减轻代谢负担，以恢复细胞生长并促进生产。为了实现代谢平衡，通过多变量模块化代谢工程精确调节生物合成途径。最终，在5升生物反应器中，T5OAc的产量提高到10.9毫克/升。与原始菌株相比，产量提高了约272倍，这是大肠杆菌中T5OAc有记录以来的最高产量，有望推动紫杉醇生物合成的进展。
