The main metabolic challenge for methylotrophs is the assimilation of single carbon units into biomass. Through de novo synthesis, methylotrophs must form carbon-carbon bonds between 1-Carbon () molecules. This is an energy intensive process, which facultative methylotrophs avoid by using a range of larger organic compounds. However, obligate methylotrophs must assimilate  molecules. There are four distinct assimilation pathways with the common theme of generating one  molecule. Bacteria use three of these pathways while Fungi use one. All four pathways incorporate 3  molecules into multi-carbon intermediates, then cleave one intermediate into a new  molecule. The remaining intermediates are rearranged to regenerate the original multi-carbon intermediates.