Converting Bacillus subtilis 168 to a Synthetic Methylotroph by Combinatorial Metabolic Regulation Strategies

Methanol, which can come from methane or carbon dioxide, is a valuable renewable one-carbon (C1) feedstock for the production of biofuels and food chemicals. A new method was developed to create a multienzyme complex by combining methanol dehydrogenase (Mdh), 3-hexulose-6-phosphate synthase (Hps), and 6-phospho-3-hexuloseisomerase (Phi) in equal parts using SpyTag/Catcher and DogTag/Catcher systems. This self-assembly of multiple enzymes improves the conversion of methanol to fructose-6-phosphate (F6P) and was used to engineer a synthetic methylotroph from B. subtilis 168 that could efficiently utilize methanol. Various metabolic regulations related to key carbon pathways were tested and integrated to boost methanol consumption in this engineered strain. The final strain, B. subtilis SM6, could consume 3.87 g/L of methanol, marking the highest level of coutilization with xylose to date. The strategies employed in this research optimized the distribution of metabolic flow for formaldehyde and xylose, offering valuable insights for future studies on synthetic methylotrophs.