Molecular, Biochemical, and Functional Characterization of a Nudix Hydrolase Protein That Stimulates the Activity of a Nicotinoprotein Alcohol Dehydrogenase

Kloosterman, Harm; Vrijbloed, Jan W.; Dijkhuizen, Lubbert

doi:10.1074/jbc.m205617200

Cited by 21 publications

(43 citation statements)

References 29 publications

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“…The results of these experiments (Table 3) show that transcription of hps and phi is induced approximately sixfold upon methanol growth. act transcription is not affected by the C source, which is contradictory to previous reports stating that mdh and act expression is under coordinated and methanol-induced control in B. methanolicus strain C1 (16). The reason for this discrepancy is unknown.…”

Section: Resultscontrasting

confidence: 55%

“…The MDH activator gene act has also been described (16), and we proceeded to analyze the regulation of these three chromosomal genes by RT-PCR as described above. The results of these experiments (Table 3) show that transcription of hps and phi is induced approximately sixfold upon methanol growth.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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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

et al. 2006

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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...

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Section: Resultscontrasting

confidence: 55%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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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

et al. 2006

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“…The gene encoding the MDH activator protein ACT was not found on pBM19, yet expression of this protein and expression of MDH in B. methanolicus have been reported to be regulated coordinately (19,20). This suggests that methanol oxidation in B. methanolicus may be governed by the concerted action of both chromosomally and plasmid-borne genes.…”

Section: Discussionmentioning

confidence: 92%

“…In B. methanolicus C1 (ϭ NCIMB 13114) it has been unequivocally demonstrated that methanol oxidation is catalyzed by the NAD(P)-dependent MDH encoded by mdh (4,15). Despite the extensive biochemical characterization of this protein (15,19,20), the mdh gene has never been reported to originate on a plasmid. The failure to complement growth of MGA3C-A6 on methanol by introducing the MDH expression plasmid pTB1.9mdhL implies that additional pBM19 genes are required for methanol consumption.…”

Section: Discussionmentioning

confidence: 99%

Plasmid-Dependent Methylotrophy in Thermotolerant Bacillus methanolicus

et al. 2004

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Bacillus methanolicus can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of 50°C. With the exception of mannitol, no sugars have been reported to support rapid growth of this organism, which is classified as a restrictive methylotroph. Here we describe the DNA sequence and characterization of a 19,167-bp circular plasmid, designated pBM19, isolated from B. methanolicus MGA3. Sequence analysis of pBM19 demonstrated the presence of the methanol dehydrogenase gene, mdh, which is crucial for methanol consumption in this bacterium. In addition, five genes (pfk, encoding phosphofructokinase; rpe, encoding ribulose-5-phosphate 3-epimerase; tkt, encoding transketolase; glpX, encoding fructose-1,6-bisphosphatase; and fba, encoding fructose-1,6-bisphosphate aldolase) with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19. A shuttle vector, pTB1.9, harboring the pBM19 minimal replicon (repB and ori) was constructed and used to transform MGA3. Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not able to grow on methanol. A pTB1.9 derivative harboring the complete mdh gene could not restore growth on methanol when it was introduced into the pBM19-cured strain, suggesting that additional pBM19 genes are required for consumption of this carbon source. Screening of 13 thermotolerant B. methanolicus wild-type strains showed that they all harbor plasmids similar to pBM19, and this is the first report describing plasmid-linked methylotrophy in any microorganism. Our findings should have an effect on future genetic manipulations of this organism, and they contribute to a new understanding of the biology of methylotrophs.

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Development of a formaldehyde biosensor with application to synthetic methylotrophy

Woolston

Roth

Kohale

et al. 2017

Biotech & Bioengineering

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Formaldehyde is a prevalent environmental toxin and a key intermediate in single carbon metabolism. The ability to monitor formaldehyde concentration is, therefore, of interest for both environmental monitoring and for metabolic engineering of native and synthetic methylotrophs, but current methods suffer from low sensitivity, complex workflows, or require expensive analytical equipment. Here we develop a formaldehyde biosensor based on the FrmR repressor protein and cognate promoter of Escherichia coli. Optimization of the native repressor binding site and regulatory architecture enabled detection at levels as low as 1 µM. We then used the sensor to benchmark the in vivo activity of several NAD-dependent methanol dehydrogenase (Mdh) variants, the rate-limiting enzyme that catalyzes the first step of methanol assimilation. In order to use this biosensor to distinguish individuals in a mixed population of Mdh variants, we developed a strategy to prevent cross-talk by using glutathione as a formaldehyde sink to minimize intercellular formaldehyde diffusion.Finally, we applied this biosensor to balance expression of mdh and the formaldehyde assimilation enzymes hps and phi in an engineered E. coli strain to minimize formaldehyde build-up while also reducing the burden of heterologous expression.This biosensor offers a quick and simple method for sensitively detecting formaldehyde, and has the potential to be used as the basis for directed evolution of Mdh and dynamic formaldehyde control strategies for establishing synthetic methylotrophy. pastoris convert methanol to formaldehyde using an FAD-linked alcohol oxidase (AOX) (Cregg, Madden, Barringer, Thill, & Stillman, 1989), and Gram-positive methylotrophs typified by Bacillus methanolicus perform the same conversion using an NAD-linked Mdh (Müller, Heggeset, Wendisch, Vorholt, & Brautaset, 2015).In all these organisms, formaldehyde acts as a branch point between further oxidization to CO 2 for energy conservation, and incorporation into biomass via the serine cycle, ribulose monophosphate (RuMP) pathway, or the xylulose-5-phosphate (Xu5P) pathway (Chistoserdova et al., 2009;Müller, Heggeset, et al., 2015;Yurimoto, Oku, & Sakai, 2011). These pathways are of growing interest in the field of metabolic engineering, where researchers seek to convert relatively cheap methanol feedstocks into higher value commodity chemicals with either native or "synthetic" methylotrophs (Kalyuzhnaya, Puri, & Lidstrom, 2015;Whitaker, Sandoval, Bennett, Fast, & Papoutsakis, 2015). Besides methylotrophs, formaldehyde is present at low levels in all organisms as a result of demethylation reactions (Kalasz, 2003). Because of its cytotoxicity, the intracellular formaldehyde concentration must be tightly controlled, which has led to the evolution of a variety of highly coordinated metabolic strategies for detoxifying formaldehyde (Yurimoto, Kato, & Sakai, 2005). The need to keep the concentration of formaldehyde low while supporting high flux places an even more stringent burden on methylotrop...

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Molecular, Biochemical, and Functional Characterization of a Nudix Hydrolase Protein That Stimulates the Activity of a Nicotinoprotein Alcohol Dehydrogenase

Cited by 21 publications

References 29 publications

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

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

Plasmid-Dependent Methylotrophy in Thermotolerant Bacillus methanolicus

Development of a formaldehyde biosensor with application to synthetic methylotrophy

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