| Bacillus methanolicus is a Gram-positive aerobic methylotroph growing optimally at 50-53 °C. Wild-type strains of B. methanolicus have been reported to secrete 58 g/l of Lglutamate in fed-batch cultures. Mutants of B. methanolicus created via classical mutangenesis can secrete 37 g/l of L-lysine, at 50 °C. The genes required for methylotrophyin B. methanolicus are encoded by an endogenous plasmid, pBM19 in strain MGA3, except for hexulose phosphate synthase (hps ) and phosphohexuloisomerase (phi) which are encoded on the chromosome. It is a promising candidate for industrial production of chemical intermediates or amino acids from methanol. B. methanolicus employs the ribulose monophospate (RuMP) pathway to assimilate the carbon derived from the methanol, but enzymes that dissimilate carbon are not identified, although formaldehyde and formate were identified as intermediates by 13C NMR. It is important to understand how methanol is oxidized to formaldehyde and then, to formate and carbon dioxide. This study aims to elucidate the methanol dissimilation pathway of B. methanolicus..;Growth rates of B. methanolicus MGA3 were assessed on methanol, mannitol, and glucose as a substrate. B. methanolicus achieved maximum growth rate, mumax, when growing on 25 mM methanol, 0.65+/-0.007 h-1, and it gradually decreased to 0.231+/-0.004 h-1 at 2Mmethanol concentration which demonstrates substrate inhibition. The maximum growth rates (mumax) of B. methanolicus MGA3 on mannitol and glucose are 0.532+/-0.002 and 0.336+/-0.003 h-1, respectively. Spiking with 100 mM methanol did not cause any growth perturbation ofB. methanolicus MGA3 when grown on methanol, whereas, 50 mM methanol was enough to halt the growth when it was grown on mannitol. However, growth of B. methanolicus MGA3 on methanol was halted completely by a 1 mM formaldehyde spike, while mannitol grown B. methanolicus MGA3 can tolerate 2 mM formaldehyde addition. Formate did not have any apparent growth perturbations at tested levels (up to 2 mM).;B. methanolicus MGA3 has a small genome, ∼3.3 Mb, and contains 2,773 ORFs larger than 100 bp. Its genome encodes two methanol dehydrogenase(mdh) genes beside the mdh on pBM19. All of the RuMP pathway genes encoded on the pBM19 have counterparts encoded on the chromosome also. The genome contains putative tetrahydrofolate (THF)- linked enzymes for formaldehyde oxidation besides putative aldehyde dehydrogenase(aldh) genes. Two putative formate dehydrogenase (fdh) genes were also identified in the genome as were the genes of the Wood-Ljungdahl pathway genes.B. methanolicus strain PB1 was also found to carry a plasmid which we named as pBM20. The difference in the formaldehyde dissimilation pathway of strain PB1 was shown via Southern Blot. The PB1 strain also has 2-fold higher formaldehyde dehydrogenase and 3-fold higher formate dehydrogenase activity compared to strain MGA3.;The methanol dissimilation pathway genes,- putative THF-linked formaldehyde oxidation genes, putative aldh genes, putative fdh gene- are all down regulated in response to spikes of methanol, formaldehyde and formate. However, the regulation of RuMP pathway genes were different in methanol vs. mannitol grown B. methanolicus MGA3 in that they were down regulated from already high levels in methanol grown cells whereas they were. |
