A metabolizable inducer-based carbon-source autoinduction (MICA) system enables low-cost, high-level protein...

Bacillus subtilis is widely used for industrial protein production and metabolic synthesis owing to its robustness, safety, and strong secretion capacity. However, high-cost and cytotoxic chemical inducers remain major bottlenecks for large-scale bioprocesses. Here, 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 promoter engineering substantially enhanced the expression strengths of all six regulatory modules. Second, integration of the T7 RNA polymerase system further amplified their output capacities, increased induction sensitivity, and reduced inducer consumption. Third, by exploiting glucose-mediated carbon catabolite repression (CCR), the maltose and mannose systems were 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 β-1,3-glucanase, as well as dynamic regulation of lycopene and β-carotene biosynthesis. Notably, the best-performing variant P_T7malA1 produced 82.5 mg L^-1 of lycopene and 94.2 mg L^-1 of β-carotene in shake-flask, representing 4.5-fold and 6.3-fold improvement over P_grac control, respectively. Upon scaling up to a 3-L bioreactor, the titers of β-carotene further reached 244.7 mg L^-1, which represent the highest levels 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.