The formaldehyde-fixing enzymes, 3-Hexulose6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), are the key enzymes catalyzing sequential reactions in the ribulose monophosphate (RuMP) pathway. In this study, we generated two fused gene constructs of the hps and phi genes (i.e.,hps-phi and phi-hps) from a methylotrophic bacterium Mycobacterium gastri MB19. The gene product of hps-phi exhibited both HPS and PHI activities at room temperature and catalyzed the sequential reactions more efficiently than a simple mixture of the individual enzymes. The gene product of phi-hps failed to display any enzyme activity. Escherichia coli strains harboring the hps-phi gene consumed formaldehyde more efficiently and exhibited better growth in a formaldehyde-containing medium than the host strain. Our results demonstrate that the engineered fusion gene has the possibility to be used to establish a formaldehyde-resistance detoxification system in various organisms.