Zhang Wei, Yang Feng, Yue Shiqiang, Lu Fuping, Mao Shuhong, Qin Hui-Min
Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education; Tianjin Key Laboratory of Industrial Microbiology; College of Biotechnology, National Engineering Laboratory for Industrial Enzymes, Tianjin University of Science and Technology, Tianjin 300457, P. R. China.
J Agric Food Chem. 2025 Jul 16;73(28):17845-17854. doi: 10.1021/acs.jafc.5c05554. Epub 2025 Jul 3.
d-Allulose, biosynthesized through D-fructose isomerization, was limited by thermodynamic equilibrium, resulting in low yields. Herein, we redesigned a redox-driven cascade pathway for d-allulose biosynthesis in single-cell systems, utilizing post-translational protein-level reprogramming tools (protease-based programmable OFF/ON-switch toolbox) to resolve enzyme incompatibility. This OFF/ON-switch was systematically optimized by adjusting the expression levels of -Lon, degron, and repressors, enabling the simultaneous switching of protein abundance to desired states within 2 h based on a thermosensitive regulator. To link the redox-driven two-step reactions to synthesize d-allulose, KEase, RDH, and FDH were placed under the control of the OFF-switch, while ADH and NOX were regulated using the ON-switch. The engineered strain T3 with the constructed OFF-ON modules achieved a d-allulose titer of 190.7 g/L with a conversion rate of 95.4%, overcoming the obstacle of thermodynamic equilibrium. This study provided a practical toolbox for rewiring reverse carbon flows by controlling enzyme levels, exemplifying the versatility of programmable protein switches.
通过D-果糖异构化生物合成的D-阿洛酮糖受热力学平衡限制，产量较低。在此，我们在单细胞系统中重新设计了一条用于D-阿洛酮糖生物合成的氧化还原驱动级联途径，利用翻译后蛋白质水平重编程工具（基于蛋白酶的可编程开/关切换工具箱）来解决酶的不相容性问题。通过调节-Lon、降解标签和阻遏物的表达水平，对这种开/关切换进行了系统优化，基于热敏调节器能够在2小时内将蛋白质丰度同时切换到所需状态。为了将氧化还原驱动的两步反应连接起来以合成D-阿洛酮糖，将酮糖异构酶（KEase）、还原酶（RDH）和甲酸脱氢酶（FDH）置于关开关的控制之下，而乙醇脱氢酶（ADH）和氧化酶（NOX）则使用开开关进行调控。构建了开-关模块的工程菌株T3实现了190.7 g/L的D-阿洛酮糖滴度，转化率为95.4%，克服了热力学平衡的障碍。本研究提供了一个通过控制酶水平来重新布线反向碳流的实用工具箱，例证了可编程蛋白质开关的多功能性。
