Enabling genetic manipulation and robustness of Bacillus methanolicus for methanol-based bio-manufacturing

Methanol-based biomanufacturing holds great promise for sustainability but is currently limited by the slow growth and low efficiency of natural or synthetic methylotrophic strains. In contrast, the thermophilic methylotroph Bacillus methanolicus exhibits rapid growth, high-temperatures tolerance, and efficient methanol utilization in defined mineral medium, making it a promising candidate for industrial applications. However, its potential is constrained by reluctant genetic modification and suboptimal robustness under fluctuating methanol concentrations. To address these limitations, we developed a comprehensive genetic manipulation system that includes an improved transformation approach, a homologous recombination-based knock-out/knock-in method, a constitutive promoter library spanning a 600-fold range of strengths, and an stringent xylose-inducible promoter with a wide dynamic range. Using these enabling tools, we enhanced the robustness of B. methanolicus under varying methanol concentrations by introducing a xylose pathway, which buffered intracellular formaldehyde accumulation. Co-utilization of methanol and xylose achieved a molar consumption ratio exceeding 4:1, indicating methanol served as the primary carbon source while xylose was auxiliary to enhance robustness. Subsequently, we developed a riboflavin cell factory by systemic engineering of B. methanolicus, achieving 2579 mg/L production in a 5-L bioreactor-the highest riboflavin titer reported for methanol-based production. This study establishes B. methanolicus as a versatile and accessible platform for sustainable methanol-based bio-manufacturing.