The MICA system

we developed the Metabolizable Inducer–based Carbon-source Autoinduction (MICA) system, which leverages six endogenous, inexpensive, non-toxic, and metabolizable carbon-source responsive regulatory modules in B. subtilis—maltose, mannose, sorbitol, arabinose, xylose, and glycerol. First, systematic engineering of key promoter elements substantially enhanced the expression strengths of all six regulatory modules. Second, integration with T7 RNA polymerase further amplified their output capacities, increased induction sensitivity, and reduced inducer consumption. Third, by exploiting glucose-mediated carbon catabolite regulation, each system was endowed with programmable autoinduction behavior, enabling induction at tunable time points through simple adjustment of glucose concentrations. Finally, the MICA system was applied to high-level production of alkaline protease and glucanase, as well as dynamic regulation of lycopene and β-carotene biosynthesis. Notably, the best-performing MICA module PT7malA1 produced 82.54 mg L⁻¹ lycopene (4.50-fold higher than Pgrac) and 94.25 mg L⁻¹ β-carotene (6.31-fold improvement than Pgrac). The β-carotene titer achieved by PT7malA1 is also the highest reported in B. subtilis to date. Collectively, the MICA system provides a versatile, low-cost, and non-toxic platform for robust protein overexpression and dynamic metabolic regulation, offering strong potential for scalable industrial biotechnology applications.

我们开发了基于代谢诱导物的碳源自动诱导（Metabolizable Inducer–based Carbon-source Autoinduction, MICA）系统，该系统依托枯草芽孢杆菌（Bacillus subtilis，下称B. subtilis）中6种内源性、低成本、无毒且可代谢的碳源响应调控模块，即麦芽糖、甘露糖、山梨醇、阿拉伯糖、木糖与甘油。其一，通过对关键启动子元件的系统性工程化改造，显著提升了全部6个调控模块的表达强度；其二，与T7 RNA聚合酶整合后，进一步放大了各模块的输出能力，提升了诱导灵敏度并降低了诱导物消耗；其三，利用葡萄糖介导的碳分解代谢物阻遏调控机制，赋予各系统可编程的自动诱导行为，可通过简单调整葡萄糖浓度实现诱导时机的精准调控；最后，将MICA系统应用于碱性蛋白酶与葡聚糖酶的高水平生产，以及番茄红素与β-胡萝卜素生物合成的动态调控。值得注意的是，性能最优的MICA模块PT7malA1可合成82.54 mg·L⁻¹番茄红素（较Pgrac提升4.50倍）与94.25 mg·L⁻¹β-胡萝卜素（较Pgrac提升6.31倍）。PT7malA1实现的β-胡萝卜素效价，亦是目前枯草芽孢杆菌中已报道的最高水平。综上，MICA系统为稳定高效的蛋白质过量表达与动态代谢调控提供了多功能、低成本且无毒的平台，在规模化工业生物技术应用中具备显著潜力。