S85: Investigations of carbon assimilation by Bacillus methanolicus MGA3 grown aerobically at 50ºC reveal new approaches for metabolic engineering as an industrial biocatalyst for amino acid and thermostable protein production from

Bacillus methanolicus, a gram-positive restrictive methylotroph, grows at 50-53 °C on methanol (MeOH) or mannitol in fresh or seawater. Wild-type strains secrete L-glutamate (58 g/l) while non-GMO auxotrophs secrete L-lysine (37 g/l) in fed-batch cultures. The genome of B. methanolicus MGA3 revealed two methanol dehydrogenase (mdh) genes in addition to the mdh on endogenous pBM19. Putative genes for aldehyde (aldh) and formate dehydrogenases (fdh), THF- and THMPT-linked C₁ dissimilation enzymes are also present suggesting multiple pathways of formaldehyde dissimilation. Quantitative qPCR spiking experiments [formaldehyde (2mM), formate (2mM) or methanol (100mM)] show C₁ assimilation and dissimilation enzymes are down-regulated when compared between MGA3 grown on MeOH and mannitol.  Expression of mdh was higher in mannitol-grown MGA3, but down-regulated when spiked with MeOH.  This suggests that MeOH metabolism is regulated by a global stress response which has significant implications for MeOH feeding of high cell density fed-batch processes. One gene up-regulated by MeOH-spiking was pfk and its promoter may be useful for MeOH-regulated expression.  Thermostable forms of MDH, ALDH and FDH have been cloned.   Surprisingly, enzymes of the Wood-Ljungdahl pathway are also present.  We report the global regulation of MeOH assimilation and dissimilation genes in response to MeOH, formaldehyde and formate, the cloning of thermostable ALDH, FDH genes.  These results suggest new approaches to engineer efficient C₁ assimilation.