Genome Sequence of Thermotolerant Bacillus methanolicus: Features and Regulation Related to Methylotrophy and Production of l -Lysine and l -Glutamate from Methanol

Abstract

bBacillus methanolicus can utilize methanol as its sole carbon and energy source, and the scientific interest in this thermotolerant bacterium has focused largely on exploring its potential as a biocatalyst for the conversion of methanol into L-lysine and L-glutamate. We present here the genome sequences of the important B. methanolicus model strain MGA3 (ATCC 53907) and the alternative wild-type strain PB1 (NCIMB13113). The physiological diversity of these two strains was demonstrated by a comparative fed-batch methanol cultivation displaying highly different methanol consumption and respiration profiles, as well as major differences in their L-glutamate production levels (406 mmol liter ؊1 and 11 mmol liter ؊1 , respectively). Both genomes are small (ca 3.4 Mbp) compared to those of other related bacilli, and MGA3 has two plasmids (pBM19 and pBM69), while PB1 has only one (pBM20). In particular, we focus here on genes representing biochemical pathways for methanol oxidation and concomitant formaldehyde assimilation and dissimilation, the important phosphoenol pyruvate/pyruvate anaplerotic node, the tricarboxylic acid cycle including the glyoxylate pathway, and the biosynthetic pathways for L-lysine and L-glutamate. Several unique findings were made, including the discovery of three different methanol dehydrogenase genes in each of the two B. methanolicus strains, and the genomic analyses were accompanied by gene expression studies. Our results provide new insight into a number of peculiar physiological and metabolic traits of B. methanolicus and open up possibilities for system-level metabolic engineering of this bacterium for the production of amino acids and other useful compounds from methanol. Methylotrophic microorganisms can utilize one-carbon (C 1 ) sources, such as methane and methanol, as their sole sources for energy and biomass generation, and there exist a variety of different enzymes and pathways for C 1 metabolism among methylotrophs (14,15). Bacteria that fix formaldehyde by the ribulose monophosphate (RuMP) pathway belong to three groups: Gramnegative obligate methylotrophs, Gram-positive facultative methylotrophs, and thermotolerant bacilli (3,4,19,38). A number of Gram-positive thermotolerant bacilli with the ability to grow on methanol at temperatures up to 60°C have been isolated, and they were later collectively classified as Bacillus methanolicus (for a review, see reference 11). B. methanolicus is a so-called restricted methylotroph, which means that it can utilize few multicarbon sources for energy and growth. The scientific interest of these organisms has mainly been dedicated to exploring their potential as cell factories for industrial production of L-lysine and L-glutamate from methanol at elevated temperatures. B. methanolicus MGA3 (ATCC 53907) was isolated from soil samples in Minnesota (38), and it has been the major model strain used for metabolic engineering of this bacterium (9,11,27).B. methanolicus has several additional unique traits, including (i) a novel NAD-dependent met...

Characterization of Fructose 1,6-Bisphosphatase and Sedoheptulose 1,7-Bisphosphatase from the Facultative Ribulose Monophosphate Cycle Methylotroph Bacillus methanolicus

“…As in E. coli (19,52), inhibition by ATP and ADP is thought to prevent futile cycling between phosphofructokinase and FBPase during growth on glycolytic carbon sources (55 (18,19,33,42). The finding that expression of glpX C is not notably induced during a shift to growth in methanol (14,56) coincides with its function as a major FBPase and sets it apart from the SBPase/FBPase encoded by glpX P , which is induced when shifted to methanol.…”

“…Thus, the RuMP cycle operates with only FBA C , GlpX P , and TA being present. Taken together, a more complex view of methylotrophy in B. methanolicus emerged, since its genome sequence has been determined (56) and since some biochemical properties of multiple copies of methanol dehydrogenase (6) and RuMP cycle enzymes (6,14,15,62,64; this study) have been elucidated. In addition, it can be anticipated that our understanding of methylotrophic growth by B. methanolicus will be furthered in particular by omics and carbon flux analyses.…”

“…TA is encoded by the chromosomal tal gene, but tal is not induced during a shift to growth on methanol (14,56). Moreover, it remains to be shown whether tal encodes a functionally active TA.…”

Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

“…The focus was put on NAD ϩ -dependent MDHs, since PQQdependent MDHs are not suitable for expression in C. glutamicum, as this organism does not synthesize PQQ or possess any PQQ-dependent enzymes (46). B. methanolicus MGA3 is a wellknown methylotroph that harbors three NAD ϩ -dependent MDHs genes (mdh, mdh2, and mdh3) and the MDH activator protein (Act), which modulates the activity of all three MDHs (47,48). In the presence of Act and with methanol as the substrate, Mdh and Mdh3 shows the highest activity in vitro (0.5 U/mg and 0.2 U/mg, respectively), whereas Mdh2 shows the lowest catalytic activity (0.14 U/mg) (48).…”

C 1 Metabolism in Corynebacterium glutamicum: an Endogenous Pathway for Oxidation of Methanol to Carbon Dioxide

“…As indicated in the introduction, methanol could be an interesting carbon source for the microbial production of food and feed additives or fine chemicals (23). To date, only a few methylotrophic bacteria, such as Bacillus methanolicus or Methylophilus methylotrophus, were engineered for the production of amino acids from methanol (64,65). As an alternative approach, it might be possible to establish the ability to utilize methanol as a carbon source in naturally nonmethylotrophic C. glutamicum production strains by introducing suitable heterologous pathways, such as the ribulose monophosphate pathway or the serine pathway.…”