Methanol valorization into glycerol by engineering Bacillus methanolicus with thermostable enzymes and a tunable promoter library.

Glycerol serves as a versatile C3 platform chemical with broad applications spanning pharmaceuticals, personal care, food, cosmetics, and as a renewable feedstock for value-added chemicals, fuels, and materials. Methanol, a promising substrate derivable from CO2, biomass, and biogas offers a sustainable route for glycerol production. Herein, we employed the thermophilic methylotroph Bacillus methanolicus as a microbial cell factory for methanol bioconversion to glycerol. Given the poor thermolability of conventional mesophilic enzymes in the glycerol synthesis pathway at elevated temperatures, we identified and introduced thermostable glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate phosphatase. These enzymes exhibited up to 185-fold longer half-life at 50 °C than their Saccharomyces cerevisiae counterparts. To precisely regulate the heterologous pathway, we engineered a synthetic promoter library comprising 100 variants, derived from the strong constitutive promoter of the 3-hexulose-6-phosphate synthase gene, which spanned five orders of magnitude in strength. By leveraging the endogenous, methylotrophy-essential plasmid pBM19 as a stable expression vector, we optimized the expression of the thermostable enzymes using selected promoters. The final engineered strain, which is independent of antibiotics and devoid of exogenous plasmids, achieved glycerol titers of 0.62 g/L in shake flasks and 8.9 g/L in bioreactors using methanol as the sole carbon source. This work constitutes the first report of glycerol production from methanol and establishes a foundational platform for synthesizing chemicals from methanol via glycerol as a key intermediate.