Alternative Route for Glyoxylate Consumption during Growth on Two-Carbon Compounds by Methylobacterium extorquens AM1

Department of Chemical Engineering, University of Washington, Seattle, WA 98195-2180, USA.
Journal of bacteriology (Impact Factor: 2.81). 04/2010; 192(7):1813-23. DOI: 10.1128/JB.01166-09
Source: PubMed

ABSTRACT Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and multicarbon compounds. Mutants defective in a pathway involved in converting acetyl-coenzyme A (CoA) to glyoxylate (the ethylmalonyl-CoA pathway) are unable to grow on both C(1) and C(2) compounds, showing that both modes of growth have this pathway in common. However, growth on C(2) compounds via the ethylmalonyl-CoA pathway should require glyoxylate consumption via malate synthase, but a mutant lacking malyl-CoA/beta-methylmalyl-CoA lyase activity (MclA1) that is assumed to be responsible for malate synthase activity still grows on C(2) compounds. Since glyoxylate is toxic to this bacterium, it seemed likely that a system is in place to keep it from accumulating. In this study, we have addressed this question and have shown by microarray analysis, mutant analysis, metabolite measurements, and (13)C-labeling experiments that M. extorquens AM1 contains an additional malyl-CoA/beta-methylmalyl-CoA lyase (MclA2) that appears to take part in glyoxylate metabolism during growth on C(2) compounds. In addition, an alternative pathway appears to be responsible for consuming part of the glyoxylate, converting it to glycine, methylene-H(4)F, and serine. Mutants lacking either pathway have a partial defect for growth on ethylamine, while mutants lacking both pathways are unable to grow appreciably on ethylamine. Our results suggest that the malate synthase reaction is a bottleneck for growth on C(2) compounds by this bacterium, which is partially alleviated by this alternative route for glyoxylate consumption. This strategy of multiple enzymes/pathways for the consumption of a toxic intermediate reflects the metabolic versatility of this facultative methylotroph and is a model for other metabolic networks involving high flux through toxic intermediates.

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    • "The dehydrogenase gene from most aerobic organisms is devoid of redox-active centers [65] and together with the hydrogen dehydrogenase gene (EC: forms a system previously known as formate hydrogenlyase. Glyoxylate is a toxic intermediate which in humans undergoes oxalate formation [66, 67] with severe consequences for the tissues involved. The glyoxylate cycle is thought to be present in bacteria, protists, plants, fungi, and nematodes but not in other Metazoa [68]. "
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    ABSTRACT: The culture-independent strategies to study microbial diversity and function have led to a revolution in environmental genomics, enabling fundamental questions about the distribution of microbes and their influence on bioremediation to be addressed. In this research we used the expression of universal stress proteins as a probe to determine the changes in degrading microbial population from a highly toxic terephthalate wastewater to a less toxic activated sludge bioreactor. The impact of relative toxicities was significantly elaborated at the levels of genus and species. The results indicated that 23 similar prokaryotic phyla were represented in both metagenomes irrespective of their relative abundance. Furthermore, the following bacteria taxa Micromonosporaceae, Streptomyces, Cyanothece sp. PCC 7822, Alicyclobacillus acidocaldarius, Bacillus halodurans, Leuconostoc mesenteroides, Lactococcus garvieae, Brucellaceae, Ralstonia solanacearum, Verminephrobacter eiseniae, Azoarcus, Acidithiobacillus ferrooxidans, Francisella tularensis, Methanothermus fervidus, and Methanocorpusculum labreanum were represented only in the activated sludge bioreactor. These highly dynamic microbes could serve as taxonomic biomarkers for toxic thresholds related to terephthalate and its derivatives. This paper, highlights the application of universal stress proteins in metagenomics analysis. Dynamics of microbial consortium of this nature can have future in biotechnological applications in bioremediation of toxic chemicals and radionuclides.
    09/2013; 2013(2):196409. DOI:10.1155/2013/196409
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    • "This was done by setting to zero the flux from crotonyl-CoA to propionyl-CoA and by adding the ICL reaction. Malate synthase, the enzyme of the glyoxylate cycle that catalyzes the condensation of acetyl-CoA and glyoxylate into malate, was not added since M. extorquens can use a combination of two enzymes to achieve the same reaction [39], as described also in R. sphaeroids [40]. As expected, the glyoxylate cycle was essential for methanol growth and was found in all assimilatory EFMs. "
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    ABSTRACT: Methylotrophic microorganisms are playing a key role in biogeochemical processes - especially the global carbon cycle - and have gained interest for biotechnological purposes. Significant progress was made in the recent years in the biochemistry, genetics, genomics, and physiology of methylotrophic bacteria, showing that methylotrophy is much more widespread and versatile than initially assumed. Despite such progress, system-level description of the methylotrophic metabolism is currently lacking, and much remains to understand regarding the network-scale organization and properties of methylotrophy, and how the methylotrophic capacity emerges from this organization, especially in facultative organisms. In this work, we report on the integrated, system-level investigation of the metabolic network of the facultative methylotroph Methylobacterium extorquens AM1, a valuable model of methylotrophic bacteria. The genome-scale metabolic network of the bacterium was reconstructed and contains 1139 reactions and 977 metabolites. The sub-network operating upon methylotrophic growth was identified from both in silico and experimental investigations, and 13C-fluxomics was applied to measure the distribution of metabolic fluxes under such conditions. The core metabolism has a highly unusual topology, in which the unique enzymes that catalyse the key steps of C1 assimilation are tightly connected by several, large metabolic cycles (serine cycle, ethylmalonyl-CoA pathway, TCA cycle, anaplerotic processes). The entire set of reactions must operate as a unique process to achieve C1 assimilation, but was shown to be structurally fragile based on network analysis. This observation suggests that in nature a strong pressure of selection must exist to maintain the methylotrophic capability. Nevertheless, substantial substrate cycling could be measured within C2/C3/C4 inter-conversions, indicating that the metabolic network is highly versatile around a flexible backbone of central reactions that allows rapid switching to multi-carbon sources. This work emphasizes that the metabolism of M. extorquens AM1 is adapted to its lifestyle not only in terms of enzymatic equipment, but also in terms of network-level structure and regulation. It suggests that the metabolism of the bacterium has evolved both structurally and functionally to an efficient but transitory utilization of methanol. Besides, this work provides a basis for metabolic engineering to convert methanol into value-added products.
    BMC Systems Biology 11/2011; 5(1):189. DOI:10.1186/1752-0509-5-189 · 2.44 Impact Factor
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    • "In the latter case, genes for the EMCP are present in the genomes but not the genes for the serine cycle. It is interesting to point out that the EMCP functions as a cycle during growth on C1 compounds (regenerating a molecule of glyoxylate per each molecule of methylene-H 4F and each molecule of CO2 assimilated; Chistoserdova et al., 2009), while during growth on C2 compounds, it functions as a liner pathway (Erb et al., 2010; Okubo et al., 2010). Some methylotrophs, however, opt to use the glyoxylate shunt instead of the EMCP. "
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    ABSTRACT: Methylotrophy is a metabolic capability possessed by microorganisms that allows them to build biomass and to obtain energy from organic substrates containing no carbon-carbon bonds (C1 compounds, such as methane, methanol, etc.). This phenomenon in microbial physiology has been a subject of study for over 100 years, elucidating a set of well-defined enzymatic systems and pathways enabling this capability. The knowledge gained from the early genetic and genomic approaches to understanding methylotrophy pointed towards the existence of alternative enzymes/pathways for the specific metabolic goals. Different combinations of these systems in different organisms suggested that methylotrophy must be modular in its nature. More recent insights from genomic analyses, including the genomes representing novel types of methylotrophs, seem to reinforce this notion. This review integrates the new findings with the previously developed concept of modularity of methylotrophy.
    Environmental Microbiology 03/2011; 13(10):2603-22. DOI:10.1111/j.1462-2920.2011.02464.x · 6.20 Impact Factor
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