Upregulated Transcription of Plasmid and Chromosomal Ribulose Monophosphate Pathway Genes Is Critical for Methanol Assimilation Rate and Methanol Tolerance in the Methylotrophic Bacterium Bacillus methanolicus

Abstract

The natural plasmid pBM19 carries the key mdh gene needed for the oxidation of methanol into formaldehyde by Bacillus methanolicus. Five more genes, glpX, fba, tkt, pfk, and rpe, with deduced roles in the cell primary metabolism, are also located on this plasmid. By using real-time PCR, we show that they are transcriptionally upregulated (6-to 40-fold) in cells utilizing methanol; a similar induction was shown for two chromosomal genes, hps and phi. These seven genes are involved in the fructose bisphosphate aldolase/sedoheptulose bisphosphatase variant of the ribulose monophosphate (RuMP) pathway for formaldehyde assimilation. Curing of pBM19 causes higher methanol tolerance and reduced formaldehyde tolerance, and the methanol tolerance is reversed to wild-type levels by reintroducing mdh. Thus, the RuMP pathway is needed to detoxify the formaldehyde produced by the methanol dehydrogenase-mediated conversion of methanol, and the in vivo transcription levels of mdh and the RuMP pathway genes reflect the methanol tolerance level of the cells. The transcriptional inducer of hps and phi genes is formaldehyde, and not methanol, and introduction of multiple copies of these two genes into B. methanolicus made the cells more tolerant of growth on high methanol concentrations. The recombinant strain also had a significantly higher specific growth rate on methanol than the wild type. While pBM19 is critical for growth on methanol and important for formaldehyde detoxification, the maintenance of this plasmid represents a burden for B. methanolicus when growing on mannitol. Our data contribute to a new and fundamental understanding of the regulation of B. methanolicus methylotrophy.Aerobic methylotrophs are bacteria capable of utilizing reduced one-carbon (C 1 ) compounds as the sole carbon source for growth and energy (2), and the majority of research on these bacteria has focused on their biochemical novelty and commercial viability. A number of gram-positive and thermotolerant Bacillus strains have been isolated and designated Bacillus methanolicus (4,26). This methylotrophic bacterium has a novel NAD-dependent methanol dehydrogenase (MDH), which contains bound NAD, to oxidize methanol into formaldehyde (11). The enzyme has a remarkably high affinity for methanol, and an activator protein called ACT modulates its in vivo activity. ACT activates MDH by hydrolysis, and the expression of these two proteins is reported to be under coordinate (and methanol-induced) control in B. methanolicus (16). Formaldehyde is the key intermediate in C 1 metabolism and can be assimilated via the ribulose monophosphate (RuMP) pathway (Fig. 1). Many RuMP pathway variants are described in the literature, and thermotolerant Bacillus strains are reported to use the fructosebisphosphate aldolase/transaldolase variant (3,4,12).Steady-state cultures of B. methanolicus MGA3 limited by methanol in the feed display sensitivity to minor methanol pulses and respond by a transient decline in biomass concentration. By using 13 C nuclear magnetic resonan...

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

“…In methylotrophs, the irreversible conversion of formaldehyde by Hps and Phi is the driving force behind shifting the pathway equilibrium toward F6P formation. In B. methanolicus, it has been suggested that the level of Hps/Phi can impact methanol consumption and cell growth (18), and the stoichiometric effect of Mdh to Hps:Phi on the methanol consumption pathway remains unexplored either in native or synthetic methylotrophs. To investigate this issue, purified Mdh3 was mixed with different ratios of a highly active Hps from M. gastri (30) to create a kinetic trap to consume formaldehyde and increase H6P production.…”

“…Detailed kinetics analyses revealed that the reverse formaldehyde reductase activity for Mdh is at least 1,000-fold higher than methanol oxidation (9), and the irreversible sequestration of formaldehyde by Hps and Phi serves as a driving force to pull the pathway equilibrium toward the desired direction. In B. methanolicus MGA3, expression of both hps and phi is more highly up-regulated than mdh upon methanol addition, and carefully coordinated expression of mdh and the RuMP pathway genes is required for efficient methanol utilization (18). In the same organism, overexpression of hps and phi results in a higher growth rate on methanol, again indicating the importance of direct coupling between the reversible methanol oxidation and the irreversible formaldehyde assimilation (18).…”

“…In B. methanolicus MGA3, expression of both hps and phi is more highly up-regulated than mdh upon methanol addition, and carefully coordinated expression of mdh and the RuMP pathway genes is required for efficient methanol utilization (18). In the same organism, overexpression of hps and phi results in a higher growth rate on methanol, again indicating the importance of direct coupling between the reversible methanol oxidation and the irreversible formaldehyde assimilation (18).…”

The Presence of N-Terminal Secretion Signal Sequences Leads to Strong Stimulation of the Total Expression Levels of Three Tested Medically Important Proteins during High-Cell-Density Cultivations of Escherichia coli

“…The experiments hereunder were performed essentially as described previously (9). Cell samples (0.1 ml) were harvested from the HCDC fermentations at an OD 600 of 130 to 160, diluted in fresh medium to an OD 600 of 10, and immediately treated with RNA Protect (QIAGEN).…”

Identification and functional characterization of a gene for the methanol : N,N'-dimethyl-4-nitrosoaniline oxidoreductase from Mycobacterium sp. strain JC1 (DSM 3803)

“…On the other hand, very little is known about the physiological role and regulation of the genes for the key enzymes responsible for the oxidation of methanol in Gram-positive methylotrophic bacteria. Only the genes for NAD-dependent MDHs of B. methanolicus strains C1 (de Vries et al, 1992) and MGA3 (Brautaset et al, 2004) have been cloned and sequenced, and several genes, including the gene for MDH, involved in the methylotrophy of B. methanolicus MGA3 were identified to be transcriptionally upregulated in cells growing with methanol (Jakobsen et al, 2006).…”