Characterization and nucleotide sequence of pqqE and pqqF in Methylobacterium extorquens AM1.
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SUBMITTER: Springer AL
PROVIDER: S-EPMC177920 | biostudies-other | 1996 Apr
REPOSITORIES: biostudies-other
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Project description:The goal of this study was to use microarrays to identify genes differentially regulated under conditions of formaldehyde stress relative to two other stress conditions (oxidative, osmotic) in an effort to identify genes that might be involved in a formaldehyde-specific stress response, rather than a general stress response, in the model methylotroph Methylobacterium extorquens AM1.
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Project description:Most serine cycle methylotrophic bacteria lack isocitrate lyase and convert acetyl coenzyme A (acetyl-CoA) to glyoxylate via a novel pathway thought to involve butyryl-CoA and propionyl-CoA as intermediates. In this study we have used a genome analysis approach followed by mutation to test a number of genes for involvement in this novel pathway. We show that methylmalonyl-CoA mutase, an R-specific crotonase, isobutyryl-CoA dehydrogenase, and a GTPase are involved in glyoxylate regeneration. We also monitored the fate of (14)C-labeled carbon originating from acetate, butyrate, or bicarbonate in mutants defective in glyoxylate regeneration and identified new potential intermediates in the pathway: ethylmalonyl-CoA, methylsuccinyl-CoA, isobutyryl-CoA, methacrylyl-CoA, and beta-hydroxyisobutyryl-CoA. A new scheme for the pathway is proposed based on these data.
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Project description:Methylobacterium extorquens AM1 is a facultative methylotrophic Alphaproteobacterium and has been subject to intense study under pure methylotrophic as well as pure heterotrophic growth conditions in the past. Here, we investigated the metabolism of M. extorquens AM1 under mixed substrate conditions, i.e., in the presence of methanol plus succinate. We found that both substrates were co-consumed, and the carbon conversion was two-thirds from succinate and one-third from methanol relative to mol carbon. (13)C-methanol labeling and liquid chromatography mass spectrometry analyses revealed the different fates of the carbon from the two substrates. Methanol was primarily oxidized to CO(2) for energy generation. However, a portion of the methanol entered biosynthetic reactions via reactions specific to the one-carbon carrier tetrahydrofolate. In contrast, succinate was primarily used to provide precursor metabolites for bulk biomass production. This work opens new perspectives on the role of methylotrophy when substrates are simultaneously available, a situation prevailing under environmental conditions.
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Project description:BackgroundButanol is a promising next generation fuel and a bulk chemical precursor. Although clostridia are the primary industrial microbes for the fermentative production of 1-butanol, alternative engineered hosts have the potential to generate 1-butanol from alternative carbon feedstocks via synthetic metabolic pathways. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, is a model system for assessing the possibility of generating products such as 1-butanol from one-carbon and two-carbon feedstocks. Moreover, the core methylotrophic pathways in M. extorquens AM1 involve unusual coenzyme A (CoA)-derivative metabolites, such as crotonyl-CoA, which is a precursor for the production of 1-butanol.ResultsIn this work, we engineered a modified CoA-dependent pathway in Methylobacterium extorquens AM1 to produce 1-butanol. Engineered strains displayed different 1-butanol titers using ethylamine as a substrate. A strain overexpressing Treponema denticola trans-enoyl-CoA reductase, Clostridium acetobutylicum alcohol dehydrogenase, and native crotonase was able to generate the highest 1-butanol titer (15.2 mg l(-1)). In vitro isotopic tracing of metabolic flux and in vivo metabolite analysis showed the accumulation of butyryl-CoA, demonstrating the functionality of the synthetic pathway and identifying targets for future improvement.ConclusionsWe demonstrated the feasibility of using metabolic intermediates of the ethylmalonyl-CoA pathway in M. extorquens AM1 to generate value-added chemicals, with 1-butanol as the test case. This will not only establish the biotechnological potential of the ethylmalonyl-CoA pathway, but will also introduce M. extorquens AM1 as a potential platform to produce value-added chemicals.
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Project description:The goal of this study was to use microarrays to identify genes differentially regulated under conditions of formaldehyde stress relative to two other stress conditions (oxidative, osmotic) in an effort to identify genes that might be involved in a formaldehyde-specific stress response, rather than a general stress response, in the model methylotroph Methylobacterium extorquens AM1. Two color experiment, three treatments, three biological replicates per treatment, and two technical (dye swap) replicates per biological replicate: formaldehyde-stressed vs. unstressed cells; oxidative-stressed vs. unstressed cells; and osmotic-stressed vs. unstressed cells.
2015-01-01 | E-GEOD-15631 | biostudies-arrayexpress
Project description:Methylobacterium extorquens AM1 was used to explore the genetics of dephosphotetrahydromethanopterin (dH(4)MPT) biosynthesis. Strains with mutations in eight "archaeal-type" genes linked on the chromosome of M. extorquens AM1 were analyzed for the ability to synthesize dH(4)MPT, and six were found to be dH(4)MPT negative. Putative functions of these genes in dH(4)MPT biosynthesis are discussed.
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Project description:PhyR represents a novel alphaproteobacterial family of response regulators having a structure consisting of two domains; a predicted amino-terminal extracytoplasmic function (ECF) sigma factor-like domain and a carboxy-terminal receiver domain. PhyR was first described in Methylobacterium extorquens AM1, in which it has been shown to be essential for plant colonization, probably due to its suggested involvement in the regulation of a number of stress proteins. Here we investigated the PhyR regulon using microarray technology. We found that the PhyR regulon is rather large and that most of the 246 targets are under positive control. Mapping of transcriptional start sites revealed candidate promoters for PhyR-mediated regulation. One of these promoters, an ECF-type promoter, was identified upstream of one-third of the target genes by in silico analysis. Among the PhyR targets are genes predicted to be involved in multiple stress responses, including katE, osmC, htrA, dnaK, gloA, dps, and uvrA. The induction of these genes is consistent with our phenotypic analyses which revealed that PhyR is involved in resistance to heat shock and desiccation, as well as oxidative, UV, ethanol, and osmotic stresses, in M. extorquens AM1. The finding that PhyR is involved in the general stress response was further substantiated by the finding that carbon starvation induces protection against heat shock and that this protection is at least in part dependent on PhyR.
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Project description:BackgroundButadiene is a platform chemical used as an industrial feedstock for the manufacture of automobile tires, synthetic resins, latex and engineering plastics. Currently, butadiene is predominantly synthesized as a byproduct of ethylene production from non-renewable petroleum resources. Although the idea of biological synthesis of butadiene from sugars has been discussed in the literature, success for that goal has so far not been reported. As a model system for methanol assimilation, Methylobacterium extorquens AM1 can produce several unique metabolic intermediates for the production of value-added chemicals, including crotonyl-CoA as a potential precursor for butadiene synthesis.ResultsIn this work, we focused on constructing a metabolic pathway to convert crotonyl-CoA into crotyl diphosphate, a direct precursor of butadiene. The engineered pathway consists of three identified enzymes, a hydroxyethylthiazole kinase (THK) from Escherichia coli, an isopentenyl phosphate kinase (IPK) from Methanothermobacter thermautotrophicus and an aldehyde/alcohol dehydrogenase (ADHE2) from Clostridium acetobutylicum. The Km and kcat of THK, IPK and ADHE2 were determined as 8.35 mM and 1.24 s-1, 1.28 mM and 153.14 s-1, and 2.34 mM and 1.15 s-1 towards crotonol, crotyl monophosphate and crotonyl-CoA, respectively. Then, the activity of one of rate-limiting enzymes, THK, was optimized by random mutagenesis coupled with a developed high-throughput screening colorimetric assay. The resulting variant (THKM82V) isolated from over 3000 colonies showed 8.6-fold higher activity than wild-type, which helped increase the titer of crotyl diphosphate to 0.76 mM, corresponding to a 7.6% conversion from crotonol in the one-pot in vitro reaction. Overexpression of native ADHE2, IPK with THKM82V under a strong promoter mxaF in M. extorquens AM1 did not produce crotyl diphosphate from crotonyl-CoA, but the engineered strain did generate 0.60 μg/mL of intracellular crotyl diphosphate from exogenously supplied crotonol at mid-exponential phase.ConclusionsThese results represent the first step in producing a butadiene precursor in recombinant M. extorquens AM1. It not only demonstrates the feasibility of converting crotonol to key intermediates for butadiene biosynthesis, it also suggests future directions for improving catalytic efficiency of aldehyde/alcohol dehydrogenase to produce butadiene precursor from methanol.
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