ABSTRACTMethanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacteriumCorynebacterium glutamicumtoward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase fromBacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway ofBacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinantC. glutamicumstrain showed an average methanol consumption rate of 1.7 ± 0.3 mM/h (mean ± standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [13C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m + 1 mass isotopomers of various intracellular metabolites. In the background of aC. glutamicumΔaldΔadhEmutant being strongly impaired in its ability to oxidize formaldehyde to CO2, the m + 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineeredC. glutamicumstrains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate.
Transaldolase in Bacillus methanolicus: biochemical characterization and biological role in ribulose monophosphate cycle
