Core pathways operating during methylotrophy of Bacillus methanolicus MGA3 and induction of a bacillithiol‐dependent detoxification pathway upon formaldehyde stress

Summary Bacillus methanolicus MGA3 is a model facultative methylotroph of interest for fundamental research and biotechnological applications. Previous research uncovered a number of pathways potentially involved in one‐carbon substrate utilization. Here, we applied dynamic 13C labeling to elucidate which of these pathways operate during growth on methanol and to uncover potentially new ones. B. methanolicus MGA3 uses the assimilatory and dissimilatory ribulose monophosphate (RuMP) cycles for conversion of the central but toxic intermediate formaldehyde. Additionally, the operation of two cofactor‐dependent formaldehyde oxidation pathways with distinct roles was revealed. One is dependent on tri‐ and tetraglutamylated tetrahydrofolate (THF) and is involved in formaldehyde oxidation during growth on methanol. A second pathway was discovered that is dependent on bacillithiol, a thiol cofactor present also in other Bacilli where it is known to function in redox‐homeostasis. We show that bacillithiol‐dependent formaldehyde oxidation is activated upon an upshift in formaldehyde induced by a substrate switch from mannitol to methanol. The genes and the corresponding enzymes involved in the biosynthesis of bacillithiol were identified by heterologous production of bacillithiol in Escherichia coli. The presented results indicate metabolic plasticity of the methylotroph allowing acclimation to fluctuating intracellular formaldehyde concentrations. In this study we applied 13C labeling to elucidate pathways involved in one‐carbon utilization of Bacillus methanolicus MGA3. Conversion of the cell toxic intermediate formaldehyde is achieved via the assimilatory and dissimilatory ribulose monophosphate (RuMP) cycle. In addition two cofactor dependent oxidation pathways with distinct metabolic purposes have been revealed. One is dependent on tri‐ and tetraglutamylated tetrahydrofolate (THF) as a cofactor and is used during growth on methanol, whereas the second depends on bacillithiol (BSH) and is activated upon methanol upshift.