Methanol metabolism in thermotolerant methylotrophic Bacillus strains involving a novel catabolic NAD-dependent methanol dehydrogenase as a key enzyme

Arfman, N; Watling, Em; Clement, W; Oosterwijk, R.; Vries, G.E de; Harder, W.; Attwood, Margaret; Dijkhuizen, Lubbert

doi:10.1007/bf00409664

Cited by 97 publications

(110 citation statements)

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“…Substrate affinities of active methylotrophs in grassland soils FG and HEG 6 were in the upper range of values of purified MDH (Arfman et al, 1989;Hektor et al, 2002), and higher than those of a previous environmental study that addresses ocean surface water communities (Dixon et al, 2011). Accordingly, the specific affinity values a 0 s were lower, and n max were higher than the solely known values that have been obtained from the environment (a 0 s : 0.12 to 0.96 d À 1 ; n max : up to 24 nmol d À 1 l À 1 ) (Dixon et al, 2011).…”

Section: Discussionmentioning

confidence: 94%

“…Accordingly, the specific affinity values a 0 s were lower, and n max were higher than the solely known values that have been obtained from the environment (a 0 s : 0.12 to 0.96 d À 1 ; n max : up to 24 nmol d À 1 l À 1 ) (Dixon et al, 2011). The lowest measured K m values (3 mmol l À 1 ) of MDH are known of the Gram-negative soil methylotroph Hyphomicrobium denitrificans (Nojiri et al, 2006), whereas K m values of MDH of other soil methylotrophs can be much higher (that is, Bacillus methanolicus, K m 4200 mmol l À 1 ) (Arfman et al, 1989;Hektor et al, 2002). H. denitrificans has a PQQ MDH (encoded by MxaFI) that has been intensively studied in the model methylotroph M. extorquens AM1 (Chistoserdova et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

“…Taxa that assimilated supplemented [ 13 C]-methanol in an aerated forest soil have been identified (Radajewski et al, 2002), but current knowledge is based on pure cultures and soil experiments (Radajewski et al, 2002;Kolb, 2009a), in which methanol was supplemented in millimolar concentrations that likely do not occur in soil. Substrate affinities (that is, half-saturation constant, K m ) of methanol dehydrogenases of soil-derived methylotrophs are above 1000 nmol l À 1 (Arfman et al, 1989;Hektor et al, 2002;Nojiri et al, 2006). Recent measurements of K m (K m ; 9.3 nmol l À 1 ) in marine surface water samples (Dixon et al, 2011) suggest that unknown and low methanol concentration-adapted methylotrophs utilize methanol in marine ecosystems.…”

Section: Introductionmentioning

confidence: 99%

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Methanol oxidation by temperate soils and environmental determinants of associated methylotrophs

et al. 2012

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The role of soil methylotrophs in methanol exchange with the atmosphere has been widely overlooked. Methanol can be derived from plant polymers and be consumed by soil microbial communities. In the current study, methanol-utilizing methylotrophs of 14 aerated soils were examined to resolve their comparative diversities and capacities to utilize ambient concentrations of methanol. Abundances of cultivable methylotrophs ranged from 10 6 -10 8 g soilDW. Methanol dissimilation was measured based on conversion of supplemented 14 C-methanol, and occurred at concentrations down to 0.002 lmol methanol g soilDW À 1. Tested soils exhibited specific affinities to methanol (a 0 s ¼ 0.01 d À 1 ) that were similar to those of other environments suggesting that methylotrophs with similar affinities were present. Two deep-branching alphaproteobacterial genotypes of mch responded to the addition of ambient concentrations of methanol (p0.6 lmol methanol g soilDW) in one of these soils. Methylotroph community structures were assessed by amplicon pyrosequencing of genes of mono carbon metabolism (mxaF, mch and fae). Alphaproteobacteria-affiliated genotypes were predominant in all investigated soils, and the occurrence of novel genotypes indicated a hitherto unveiled diversity of methylotrophs. Correlations between vegetation type, soil pH and methylotroph community structure suggested that plant-methylotroph interactions were determinative for soil methylotrophs.

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Methanol oxidation by temperate soils and environmental determinants of associated methylotrophs

et al. 2012

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“…(Figure 3). Bacillus strains growing on methylamine, methanol and dimethylsulfide have been characterized (Dijkhuizen et al, 1988;Arfman et al, 1989;Anesti et al, 2005). Though the methylamine degradation pathway in the genus Bacillus is poorly characterized, all Gram-positive methylotrophs studied to date use the methylamine oxidase pathway (Chistoserdova et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

Active methylotrophs in the sediments of Lonar Lake, a saline and alkaline ecosystem formed by meteor impact

et al. 2010

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Lonar Lake is a unique saline and alkaline ecosystem formed by meteor impact in the Deccan basalts in India around 52 000 years ago. To investigate the role of methylotrophy in the cycling of carbon in this unusual environment, stable-isotope probing (SIP) was carried out using the onecarbon compounds methane, methanol and methylamine. Denaturing gradient gel electrophoresis fingerprinting analyses performed with heavy 13 C-labelled DNA retrieved from sediment microcosms confirmed the enrichment and labelling of active methylotrophic communities. Clone libraries were constructed using PCR primers targeting 16S rRNA genes and functional genes. Methylomicrobium, Methylophaga and Bacillus spp. were identified as the predominant active methylotrophs in methane, methanol and methylamine SIP microcosms, respectively. Absence of mauA gene amplification in the methylamine SIP heavy fraction also indicated that methylamine metabolism in Lonar Lake sediments may not be mediated by the methylamine dehydrogenase enzyme pathway. Many gene sequences retrieved in this study were not affiliated with extant methanotrophs or methylotrophs. These sequences may represent hitherto uncharacterized novel methylotrophs or heterotrophic organisms that may have been cross-feeding on methylotrophic metabolites or biomass. This study represents an essential first step towards understanding the relevance of methylotrophy in the soda lake sediments of an unusual impact crater structure.

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“…The ability to use methanol is initiated by its oxidation to formaldehyde by methanol dehydrogenase (Mdh) encoded by an mdh gene (8). There are two classes of Mdhs among methylotrophic bacteria: the well-studied pyrroloquinoline quinone (PQQ)-containing Mdhs, which are found in the most ubiquitous, Gram-negative, strictly aerobic methylotophs, and the NAD(P) + -dependent, cytoplasmic Mdhs commonly found in thermophilic Gram-positive bacteria (9).…”

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confidence: 99%

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

Price

Chen

Whitaker

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

104

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Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channeling is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3-ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an "NADH Sink" was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol. methane | methylotophs | supramolcular | scaffold | substrate channeling

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Methanol metabolism in thermotolerant methylotrophic Bacillus strains involving a novel catabolic NAD-dependent methanol dehydrogenase as a key enzyme

Cited by 97 publications

References 41 publications

Methanol oxidation by temperate soils and environmental determinants of associated methylotrophs

Methanol oxidation by temperate soils and environmental determinants of associated methylotrophs

Active methylotrophs in the sediments of Lonar Lake, a saline and alkaline ecosystem formed by meteor impact

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

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