Co-Consumption of Methanol and Succinate by Methylobacterium extorquens AM1

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. ¹³C-methanol labeling and liquid chromatography mass spectrometry analyses revealed the different fates of the carbon from the two substrates. Methanol was primarily oxidized to CO₂ 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.     

Methanol Assimilation in Methylobacterium extorquens AM1: Demonstration of All Enzymes and Their Regulation

Background

Methylobacterium extorquens AM1 is an aerobic facultative methylotrophic α-proteobacterium that can use reduced one-carbon compounds such as methanol, but also multi-carbon substrates like acetate (C₂) or succinate (C₄) as sole carbon and energy source. The organism has gained interest as future biotechnological production platform based on methanol as feedstock.

Methodology/Principal Findings

We present a comprehensive study of all postulated enzymes for the assimilation of methanol and their regulation in response to the carbon source. Formaldehyde, which is derived from methanol oxidation, is assimilated via the serine cycle, which starts with glyoxylate and forms acetyl-CoA. Acetyl-CoA is assimilated via the proposed ethylmalonyl-CoA pathway, which thereby regenerates glyoxylate. To further the understanding of the central carbon metabolism we identified and quantified all enzymes of the pathways involved in methanol assimilation. We observed a strict differential regulation of their activity level depending on whether C₁, C₂ or C₄ compounds are used. The enzymes, which are specifically required for the utilization of the individual substrates, were several-fold up-regulated and those not required were down-regulated. The enzymes of the ethylmalonyl-CoA pathway showed specific activities, which were higher than the calculated minimal values that can account for the observed growth rate. Yet, some enzymes of the serine cycle, notably its first and last enzymes serine hydroxymethyl transferase and malate thiokinase, exhibit much lower values and probably are rate limiting during methylotrophic growth. We identified the natural C₁ carrying coenzyme as tetrahydropteroyl-tetraglutamate rather than tetrahydrofolate.

Conclusion/Significance

This study provides the first complete picture of the enzymes required for methanol assimilation, the regulation of their activity levels in response to the growth substrate, and the identification of potential growth limiting steps.     

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

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 C₁ and C₂ compounds, showing that both modes of growth have this pathway in common. However, growth on C₂ compounds via the ethylmalonyl-CoA pathway should require glyoxylate consumption via malate synthase, but a mutant lacking malyl-CoA/β-methylmalyl-CoA lyase activity (MclA1) that is assumed to be responsible for malate synthase activity still grows on C₂ 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 ¹³C-labeling experiments that M. extorquens AM1 contains an additional malyl-CoA/β-methylmalyl-CoA lyase (MclA2) that appears to take part in glyoxylate metabolism during growth on C₂ compounds. In addition, an alternative pathway appears to be responsible for consuming part of the glyoxylate, converting it to glycine, methylene-H₄F, 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 C₂ 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.Methylobacterium extorquens AM1 grows on one-carbon (C₁) compounds using the serine cycle for assimilation (25). This metabolism requires the conversion of acetyl-coenzyme A (CoA) to glyoxylate, which occurs via a novel pathway in which acetyl-CoA is converted to methylsuccinyl-CoA via acetoacetyl-CoA, ß-hydroxybutyryl-CoA, and ethylmalonyl-CoA (30-33). Recently, the steps involved in the conversion of methylsuccinyl-CoA to glyoxylate have been elucidated, and the pathway has been termed the ethylmalonyl-CoA (EMC) pathway (1, 19, 20, 40). Careful labeling measurements coupled to measurements of intermediates has confirmed that, during the growth of M. extorquens AM1 on methanol, methylsuccinyl-CoA is converted to glyoxylate and propionyl-CoA via mesaconyl-CoA and ß-methylmalyl-CoA (40).This finding has raised questions regarding how M. extorquens AM1 grows on two-carbon (C₂) compounds. The pathway involved in the conversion of acetyl-CoA to glyoxylate is known to operate during growth on both C₁ and C₂ compounds, as mutants in genes involved in this conversion are unable to grow on either C₁ or C₂ compounds, and in both cases they are rescued by glyoxylate (11, 15-17, 44). If glyoxylate is produced as an end product of this pathway during C₂ growth, then it must be converted to an intermediate of central metabolism, which has been proposed to involve a malate synthase activity (2, 14-17) (Fig. ​(Fig.1).1). In M. extorquens AM1, the apparent malate synthase activity is carried out in two steps, first by converting acetyl-CoA and glyoxylate to malyl-CoA by malyl-CoA lyase and then by converting malyl-CoA to malate by malyl-CoA hydrolase (Fig. ​(Fig.1)1) (14). However, a mutant (PCT57) defective in malyl-CoA lyase (MclA1) (22), which contains no detectable malate synthase activity during growth on methanol, is able to grow on C₂ compounds (43).Open in a separate windowFIG. 1.Enzymes and genes involved in the ethylmalonyol-CoA pathway. The colors of gene names denote a change in gene expression from microarray results comparing wild-type cells grown on ethylamine to those grown on succinate: dark red, >3-fold increase; light red, 1.5- to 3-fold increase; black, no significant change (1.49-fold increase to 1.49-fold decrease); light green, 1.5- to 3-fold decrease; dark green, >3-fold decrease. Parentheses denote a predicted function not confirmed by the mutant phenotype. See Table ​Table11 for enzyme names.Clearly, the finding that glyoxylate is generated as a direct product of the EMC pathway presents a conundrum. Apparently acetyl-CoA is converted to glyoxylate via this pathway, but M. extorquens AM1 lacking malate synthase is able to grow on C₂ compounds. Another apparent conundrum involving the malyl-CoA lyase (mclA1) mutant is that the EMC pathway requires an enzyme that carries out ß-methylmalyl-CoA cleavage, a reaction that homologs of MclA1 are known to carry out (38). The MclA1 enzyme has been purified from M. extorquens AM1 and shown to have activity with glyoxylate and propionyl-CoA (27), which would produce ß-methylmalyl-CoA. These results have led to the suggestion that MclA homologs actually are malyl-CoA/ß-methylmalyl-CoA lyases (38). Since the mclA1 mutant does not contain detectable malyl-CoA lyase activity, and by inference has correspondingly low ß-methylmalyl-CoA lyase activity, it was not clear how M. extorquens AM1 could convert acetyl-CoA to propionyl-CoA and glyoxylate via the EMC pathway in the mclA1 mutant.The purpose of this study was to solve these conundrums and determine how mutants of M. extorquens AM1 grow on C₂ compounds in the absence of malyl-CoA/ß-methylmalyl-CoA lyase or malate synthase activity. Our results show (i) that the known homolog of MclA1 (MclA2) appears to be capable of supporting both ß-methylmalyl-CoA cleavage and condensation between glyoxylate and acetyl-CoA in the mclA1 mutant, and (ii) that an alternative route for glyoxylate consumption occurs in this bacterium, in which it is converted to intermediates of central metabolism via a part of the serine cycle coupled with the glycine cleavage system.     

Formaldehyde-detoxifying role of the tetrahydromethanopterin-linked pathway in Methylobacterium extorquens AM1

The facultative methylotroph Methylobacterium extorquens AM1 possesses two pterin-dependent pathways for C(1) transfer between formaldehyde and formate, the tetrahydrofolate (H(4)F)-linked pathway and the tetrahydromethanopterin (H(4)MPT)-linked pathway. Both pathways are required for growth on C(1) substrates; however, mutants defective for the H(4)MPT pathway reveal a unique phenotype of being inhibited by methanol during growth on multicarbon compounds such as succinate. It has been previously proposed that this methanol-sensitive phenotype is due to the inability to effectively detoxify formaldehyde produced from methanol. Here we present a comparative physiological characterization of four mutants defective in the H(4)MPT pathway and place them into three different phenotypic classes that are concordant with the biochemical roles of the respective enzymes. We demonstrate that the analogous H(4)F pathway present in M. extorquens AM1 cannot fulfill the formaldehyde detoxification function, while a heterologously expressed pathway linked to glutathione and NAD(+) can successfully substitute for the H(4)MPT pathway. Additionally, null mutants were generated in genes previously thought to be essential, indicating that the H(4)MPT pathway is not absolutely required during growth on multicarbon compounds. These results define the role of the H(4)MPT pathway as the primary formaldehyde oxidation and detoxification pathway in M. extorquens AM1.     

A Catalytic Role of XoxF1 as La3+-Dependent Methanol Dehydrogenase in Methylobacterium extorquens Strain AM1

In the methylotrophic bacterium Methylobacterium extorquens strain AM1, MxaF, a Ca²⁺-dependent methanol dehydrogenase (MDH), is the main enzyme catalyzing methanol oxidation during growth on methanol. The genome of strain AM1 contains another MDH gene homologue, xoxF1, whose function in methanol metabolism has remained unclear. In this work, we show that XoxF1 also functions as an MDH and is La³⁺-dependent. Despite the absence of Ca²⁺ in the medium strain AM1 was able to grow on methanol in the presence of La³⁺. Addition of La³⁺ increased MDH activity but the addition had no effect on mxaF or xoxF1 expression level. We purified MDH from strain AM1 grown on methanol in the presence of La³⁺, and its N-terminal amino acid sequence corresponded to that of XoxF1. The enzyme contained La³⁺ as a cofactor. The ΔmxaF mutant strain could not grow on methanol in the presence of Ca²⁺, but was able to grow after supplementation with La³⁺. Taken together, these results show that XoxF1 participates in methanol metabolism as a La³⁺-dependent MDH in strain AM1.     

Metabolite profiling analysis of Methylobacterium extorquens AM1 by comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry

Methylobacterium extorquens AM1 is a facultative methylotroph, which is a potential candidate to be used in commercial processes to convert simple one-carbon compounds to a variety of multicarbon chemicals and products. To better understand C(1) metabolism in M. extorquens AM1 at the systems level, metabolite profiling tools were developed and applied in this bacterium. Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC x GC-TOFMS) was used to obtain metabolite profiles of M. extorquens AM1 (primarily organic acids) and to identify the metabolite differences between cells grown on methanol (C(1) substrate) and succinate (multicarbon substrate). In this study, a list of compounds that included amino acids and major intermediates of central C(1) and multicarbon metabolism were studied as target metabolites. For these, calibration curves were obtained for absolute quantification by spiking different amounts of standard mixtures to cell cultures. Parallel factor analysis (PARAFAC) was used for accurate peak quantification. Unknown chemical differences between cells grown on methanol and succinate were identified by applying Fisher ratio analysis at a selective mass channel (m/z 147). Thirty-six compounds were discovered to be statistically differentially expressed between C(1) and multicarbon metabolism. Among these, 13 were identified by matching to library mass spectra, and the rest were novel compounds that were not included in libraries. These differentially expressed compounds have provided clues to new pathways that are specifically linked to C(1) metabolism.     

Towards Kinetic Modeling of Global Metabolic Networks: Methylobacterium extorquens AM1 Growth as Validation

Here we report a systematic method for constructing a large scale kinetic metabolic model and its initial application to the modeling of central metabolism of Methylobacterium extorquens AM1, a methylotrophic and environmental important bacterium. Its central metabolic network includes formaldehyde metabolism, serine cycle, citric acid cycle, pentose phosphate pathway, gluconeogensis, PHB synthesis and acetyl-CoA conversion pathway, respiration and energy metabolism. Through a systematic and consistent procedure of finding a set of parameters in the physiological range we overcome an outstanding difficulty in large scale kinetic modeling: the requirement for a massive number of enzymatic reaction parameters. We are able to construct the kinetic model based on general biological considerations and incomplete experimental kinetic parameters. Our method consists of the following major steps: 1) using a generic enzymatic rate equation to reduce the number of enzymatic parameters to a minimum set while still preserving their characteristics; 2) using a set of steady state fluxes and metabolite concentrations in the physiological range as the expected output steady state fluxes and metabolite concentrations for the kinetic model to restrict the parametric space of enzymatic reactions; 3) choosing enzyme constants K’s and K’eqs optimized for reactions under physiological concentrations, if their experimental values are unknown; 4) for models which do not cover the entire metabolic network of the organisms, designing a dynamical exchange for the coupling between the metabolism represented in the model and the rest not included.     

Towards Kinetic Modeling of Global Metabolic Networks: Methylobacterium extorquens AM1 Growth as Validation

Here we report a systematic method for constructing a large scale kinetic metabolic model and its initial application to the modeling of central metabolism of Methylobacterium extorquens AM1, a methylotrophic and environmental important bacterium. Its central metabolic network includes formaldehyde metabolism, serine cycle, citric acid cycle, pentose phosphate pathway, gluconeogensis, PHB synthesis and acetyl-CoA conversion pathway, respiration and energy metabolism. Through a systematic and consistent procedure of finding a set of parameters in the physiological range we overcome an outstanding difficulty in large scale kinetic modeling: the requirement for a massive number of enzymatic reaction parameters. We are able to construct the kinetic model based on general biological considerations and incomplete experimental kinetic parameters. Our method consists of the following major steps: 1) using a generic enzymatic rate equation to reduce the number of enzymatic parameters to a minimum set while still preserving their characteristics; 2) using a set of steady state fluxes and metabolite concentrations in the physiological range as the expected output steady state fluxes and metabolite concentrations for the kinetic model to restrict the parametric space of enzymatic reactions; 3) choosing enzyme constants K’s and K’eqs optimized for reactions under physiological concentrations, if their experimental values are unknown; 4) for models which do not cover the entire metabolic network of the organisms, designing a dynamical exchange for the coupling between the metabolism represented in the model and the rest not included.     

Novel formaldehyde-activating enzyme in Methylobacterium extorquens AM1 required for growth on methanol

Formaldehyde is toxic for all organisms from bacteria to humans due to its reactivity with biological macromolecules. Organisms that grow aerobically on single-carbon compounds such as methanol and methane face a special challenge in this regard because formaldehyde is a central metabolic intermediate during methylotrophic growth. In the alpha-proteobacterium Methylobacterium extorquens AM1, we found a previously unknown enzyme that efficiently catalyzes the removal of formaldehyde: it catalyzes the condensation of formaldehyde and tetrahydromethanopterin to methylene tetrahydromethanopterin, a reaction which also proceeds spontaneously, but at a lower rate than that of the enzyme-catalyzed reaction. Formaldehyde-activating enzyme (Fae) was purified from M. extorquens AM1 and found to be one of the major proteins in the cytoplasm. The encoding gene is located within a cluster of genes for enzymes involved in the further oxidation of methylene tetrahydromethanopterin to CO(2). Mutants of M. extorquens AM1 defective in Fae were able to grow on succinate but not on methanol and were much more sensitive toward methanol and formaldehyde. Uncharacterized orthologs to this enzyme are predicted to be encoded by uncharacterized genes from archaea, indicating that this type of enzyme occurs outside the methylotrophic bacteria.     

Physiological analysis of Methylobacterium extorquens AM1 grown in continuous and batch cultures

Chemostat cultures of Methylobacterium extorquens AM1 grown on methanol or succinate at a range of dilution rates were compared to batch cultures in terms of enzyme levels, poly-β-hydroxybutyrate content, and intracellular concentrations of adenine and pyridine nucleotides. In both chemostat and batch cultures, enzymes specific to C₁ metabolism were up-regulated during growth on methanol and down-regulated during growth on succinate, polyhydroxybutyrate levels were higher on succinate, intracellular ATP levels and the energy charge were higher during growth on methanol, while the pools of reducing equivalents were higher during growth on succinate. For most of the tested parameters, little alteration occurred in response to growth rate. Overall, we conclude that the chemostat cultivation conditions developed in this study roughly mimic the growth in batch cultures, but provide a better control over the culturing conditions and a better data reproducibility, which are important for integrative functional studies. This study provides baseline data for future work using chemostat cultures, defining key similarities and differences in the physiology compared to existing batch culture data.     

XoxF is required for expression of methanol dehydrogenase in Methylobacterium extorquens AM1

In Gram-negative methylotrophic bacteria, the first step in methylotrophic growth is the oxidation of methanol to formaldehyde in the periplasm by methanol dehydrogenase. In most organisms studied to date, this enzyme consists of the MxaF and MxaI proteins, which make up the large and small subunits of this heterotetrameric enzyme. The Methylobacterium extorquens AM1 genome contains two homologs of MxaF, XoxF1 and XoxF2, which are ～50% identical to MxaF and ～90% identical to each other. It was previously reported that xoxF is not required for methanol growth in M. extorquens AM1, but here we show that when both xoxF homologs are absent, strains are unable to grow in methanol medium and lack methanol dehydrogenase activity. We demonstrate that these defects result from the loss of gene expression from the mxa promoter and suggest that XoxF is part of a complex regulatory cascade involving the 2-component systems MxcQE and MxbDM, which are required for the expression of the methanol dehydrogenase genes.     

Characterization and nucleotide sequence of pqqE and pqqF in Methylobacterium extorquens AM1.

Methylobacterium extorquens AM1 pqqEF are genes required for synthesis of pyrroloquinoline quinone (PQQ). The nucleotide sequence of these genes indicates PqqE belongs to an endopeptidase family, including PqqF of Klebsiella pneumoniae, and M. extorquens AM1 PqqF has low identity with the same endopeptidase family. M. extorquens AM1 pqqE complemented a K. pneumoniae pqqF mutant.     

Crystallization and preliminary crystallographic investigation of methanol dehydrogenase from Methylobacterium extorquens AM1.

Single crystals of methanol dehydrogenase (MDH) from Methylobacterium extorquens AM1 have been grown by the vapour diffusion method. These crystals diffract to beyond 2 A resolution and are suitable for X-ray crystallography. They belong to the orthorhombic space group P2(1)2(1)2(1) and have the following unit cell parameters: a = 66.79 A, b = 108.9 A, c = 188.9 A. One asymmetric unit contains an alpha 2 beta 2 tetramer of MDH and the location of the non-crystallographic 2-fold symmetry axis of this tetramer is defined by the paired positions of the binding sites of heavy atoms in four MDH-derivatives.     

Comprehensive proteomics of Methylobacterium extorquens AM1 metabolism under single carbon and nonmethylotrophic conditions

In order to validate a gel free quantitative proteomics assay for the model methylotrophic bacterium Methylobacterium extorquens AM1, we examined the M. extorquens AM1 proteome under single carbon (methanol) and multicarbon (succinate) growth, conditions that have been studied for decades and for which extensive corroborative data have been compiled. In total, 4447 proteins from a database containing 7556 putative ORFs from M. extorquens AM1 could be identified with two or more peptide sequences, corresponding to a qualitative proteome coverage of 58%. Statistically significant nonzero (log(2) scale) differential abundance ratios of methanol/succinate could be detected for 317 proteins using summed ion intensity measurements and 585 proteins using spectral counting, at a q-value cut-off of 0.01, a measure of false discovery rate. The results were compared to recent microarray studies performed under equivalent chemostat conditions. The M. extorquens AM1 studies demonstrated the feasibility of scaling up the multidimensional capillary HPLC MS/MS approach to a prokaryotic organism with a proteome more than three times the size of microbes we have investigated previously, while maintaining a high degree of proteome coverage and reliable quantitative abundance ratios.