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.     

Microbial growth on oxalate by a route not involving glyoxylate carboligase

1. The metabolism of oxalate by the pink-pigmented organisms, Pseudomonas AM1, Pseudomonas AM2, Protaminobacter ruber and Pseudomonas extorquens has been compared with that of the non-pigmented Pseudomonas oxalaticus. 2. During growth on oxalate, all the organisms contain oxalyl-CoA decarboxylase, formate dehydrogenase and oxalyl-CoA reductase. This is consistent with oxidation of oxalate to carbon dioxide taking place via oxalyl-CoA, formyl-CoA and formate as intermediates, and also reduction of oxalate to glyoxylate taking place via oxalyl-CoA. 3. The pink-pigmented organisms, when grown on oxalate, contain l-serine–glyoxylate aminotransferase and hydroxypyruvate reductase but do not contain glyoxylate carboligase. The converse of this obtains in oxalate-grown Ps. oxalaticus. This indicates that, in contrast with Ps. oxalaticus, synthesis of C₃ compounds from oxalate by the pink-pigmented organisms occurs by a variant of the `serine pathway' used by Pseudomonas AM1 during growth on C₁ compounds. 4. Evidence in favour of this scheme is provided by the finding that a mutant of Pseudomonas AM1 that lacks hydroxypyruvate reductase is not able to grow on oxalate.     

Acetate utilization by a methylotrophic bacterium

The mode of acetate ultilization in the Gram-negative diplococcoid bacterium PAR was investigated. This organism uses the isocitrate lyase-negative serine pathway during growth on C₁-compounds and was found to lack isocitrate lyase on acetate growth also. Enzyme assays revealed the absence of the glycerate and hydroxyaspartate pathways for the metabolism of C₂-compounds. Pulse-labeling experiments indicated the rapid formation of glycine, glutamate, and malate. The rapid formation of malate and the presence of malate synthase suggest that glyoxylate is an intermediate formed from acetate by a means other than the conventional isocitrate cleavage reaction.     

Nitrogen assimilation in facultative methylotrophic bacteria

A study was done of the pathways of nitrogen assimilation in the facultative methylotrophsPseudomonas MA andPseudomonas AM1, with ammonia or methylamine as nitrogen sources and with methylamine or succinate as carbon sources. When methylamine was the sole carbon and/or nitrogen source, both organisms possessed enzymes of the glutamine synthetase/glutamate synthase pathway, but when ammonia was the nitrogen sourcePseudomonas AM1 also synthesized glutamate dehydrogenase with a pH optimum of 9.0, andPseudomonas MA elaborated both glutamate dehydrogenase (pH optimum 7.5) and alanine dehydrogenase (pH optimum 9.0). Glutamate dehydrogenase and glutamate synthase from both organisms were solely NADPH-dependent; alanine dehydrogenase was NADH-dependent. No evidence was obtained for regulation of glutamine synthetase by adenylylation in either organism, nor did glutamine synthetase appear to regulate glutamate dehydrogenase synthesis.     

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.     

Metabolism of dodecyldimethylamine by Pseudomonas MA3

Pseudomonas MA3 was isolated from activated sludge on the basis of its capacity to use dodecyldimethylamine as a sole carbon (C) and energy source. Dodecylamine, dodecanal, dodecanoic acid and acetic acid also supported growth of Pseudomonas MA3. Dodecyldimethylamine-grown cells oxidized a wide range of alkylamine derivatives, dodecanal, dodecanoic acid and acetic acid. Degradation of the alkyl chain of dodecyldimethylamine by Pseudomonas MA3 appeared from the stoichiometric liberation of dimethylamine. A dehydrogenase catalysed the cleavage of the C_alkyl-N bond. The first intermediate of the proposed degradation pathway, dodecanal, accumulated in the presence of decanal used as a competitive inhibitor. The second intermediate,dodecanoic acid, was formed in the presence of acrylic acid during the degradation of dodecyldimethylamine. Dodecanal was converted into dodecanoic acid by a dehydrogenase and dodecanoic acid was then degraded via the oxidation pathway.     

Biosynthesis of polyhydroxyalkanoate copolymers from methanol by Methylobacterium extorquens AM1 and the engineered strains under cobalt-deficient conditions

Methylobacterium extorquens AM1 has been shown to accumulate polyhydroxyalkanoate (PHA) composed solely of (R)-3-hydroxybutyrate (3HB) during methylotrophic growth. The present study demonstrated that the wild-type strain AM1 grown under Co²⁺-deficient conditions accumulated copolyesters of 3HB and a C₅-monomer, (R)-3-hydroxyvalerate (3HV), using methanol as the sole carbon source. The 3HV unit was supposed to be derived from propionyl-CoA, synthesized via the ethylmalonyl-CoA pathway impaired by Co²⁺ limitation. This assumption was strongly supported by the dominant incorporation of the 3HV unit into PHA when a strain lacking propionyl-CoA carboxylase was incubated with methanol. Further genetic engineering of M. extorquens AM1 was employed for the methylotrophic synthesis of PHA copolymers. A recombinant strain of M. extorquens AM1C_Ac in which the original PHA synthase gene phaC _Me had been replaced by phaC _Ac , encoding an enzyme with broad substrate specificity from Aeromonas caviae, produced a PHA terpolymer composed of 3HB, 3HV, and a C₆-monomer, (R)-3-hydroxyhexanoate, from methanol. The cellular content and molecular weight of the PHA accumulated in the strain AM1C_Ac were higher than those of PHA in the wild-type strain. The triple deletion of three PHA depolymerase genes in M. extorquens AM1C_Ac showed no significant effects on growth and PHA biosynthesis properties. Overexpression of the genes encoding β-ketothiolase and NADPH-acetoacetyl-CoA reductase increased the cellular PHA content and 3HV composition in PHA, although the cell growth on methanol was decreased. This study opens up the possibility of producing practical PHA copolymers with methylotrophic bacteria using methanol as a feedstock.     

Metabolism of threonine by Fusaria growth on threonine as the sole carbon and nitrogen source

Three strains ofFusarium supporting aerobic growth onl-threonine as the sole source of energy and carbon and nitrogen, initially metabolised threonine to acetyl-CoA and glycine via induciblel-threonine:NAD⁺ dehydrogenase plus 2-amino-3-oxobutyrate:CoA ligase activities. Comparative enzyme induction patterns after growth of the three strains on a wide range of carbon sources indicated that the glycine produced by the NAD⁺ plus CoASH-dependent cleavage of threonine was subsequently utilised as an energy source and biosynthetic precursor via the glycine-serine pathway, pyruvate carboxylase, and ultimately the TCA cycle. Acetyl-CoA, the second initial C₂ threonine catabolism product, was subsequently assimilated via a combined TCA plus glyoxylate cycle.     

Metabolic Features of Methane- and Methanol-Utilizing Bacteria

Investigations of a wide range of methane- and methanol-utilizers showed a striking versatility of their metabolism dependent on the genotype and growth conditions. A correlation between pathways of carbon and nitrogen metabolism was found. It was most stringent in obligate methane-utilizers: the hexulosephosphate pathway bacteria assimilated NH₃ by the reductive amination of α-ketoglutarate or pyruvate whereas the serine pathway bacteria used the glutamate cycle (glutamine synthetase + glutamate-oxoglutarate aminotransferase). Multiple enzymic lesions were found in central metabolism of obligate methylotrophs, i.e. the absence of the enzymes of glycolytic and pentosephosphate pathways, gluconeogenesis, citric acid cycle and glyoxylate shunt. These metabolic blocks were not so profound and could be compensated in restricted and facultative methylotrophs during heterotrophic growth. The average levels of exogenous CO₂ fixation in methylotrophic bacteria with the hexulosephosphate, serine and ribulosebisphosphate pathways were found to be 10, 30 and 80% of their total cell carbon, respectively. These results served as a basis for biotechnological applications of metabolic potential of methylotrophs (production of biomass, polysaccharides and enzymes as well as for microbiological treatment of industrial waters containing toxic C₁- and C_n-compounds).     

Expression of the sub-pathways of the Chloroflexus aurantiacus 3-hydroxypropionate carbon fixation bicycle in E. coli: Toward horizontal transfer of autotrophic growth

The 3-hydroxypropionate (3-HPA) bicycle is unique among CO₂-fixing systems in that none of its enzymes appear to be affected by oxygen. Moreover, the bicycle includes a number of enzymes that produce novel intermediates of biotechnological interest, and the CO₂-fixing steps in this pathway are relatively rapid. We expressed portions of the 3-HPA bicycle in a heterologous organism, E. coli K12. We subdivided the 3-HPA bicycle into four sub-pathways: (1) synthesis of propionyl-CoA from acetyl-CoA, (2) synthesis of succinate from propionyl-CoA, (3) glyoxylate production and regeneration of acetyl-CoA, and (4) assimilation of glyoxylate and propionyl-CoA to form pyruvate and regenerate acetyl-CoA. We expressed the novel enzymes of the 3-HPA bicycle in operon form and used phenotypic tests for activity. Sub-pathway 1 activated a propionate-specific biosensor. Sub-pathway 2, found in non-CO₂-fixing bacteria, was reassembled in E. coli using genes from diverse sources. Sub-pathway 3, operating in reverse, generated succinyl-CoA sufficient to rescue a sucAD⁻ double mutant of its diaminopimelic acid (DAP) auxotrophy. Sub-pathway 4 was able to reduce the toxicity of propionate and allow propionate to contribute to cell biomass in a prpC⁻(2 methylcitrate synthase) mutant strain. These results indicate that all of the sub-pathways of the 3-HPA bicycle can function to some extent in vivo in a heterologous organism, as indicated by growth tests. Overexpression of certain enzymes was deleterious to cell growth, and, in particular, expression of MMC-CoA lyase caused a mucoid phenotype. These results have implications for metabolic engineering and for bacterial evolution through horizontal gene transfer.     

Substrate specificity of a long-chain alkylamine-degrading <Emphasis Type="Italic">Pseudomonas</Emphasis> sp isolated from activated sludge

A bacterium strain BERT, which utilizes primary long-chain alkylamines as nitrogen, carbon and energy source, was isolated from activated sludge. This rod-shaped motile, Gram-negative strain was identified as a Pseudomonas sp. The substrate spectrum of this Pseudomonas strain BERT includes primary alkylamines with alkyl chains ranging from C₃ to C₁₈, and dodecyl-1,3-diaminopropane. Amines with alkyl chains ranging from 8 to 14 carbons were the preferred substrates. Growth on dodecanal, dodecanoic acid and acetic acid and simultaneous adaptation studies indicated that this bacterium initiates degradation through a C_alkyl–N cleavage. The cleavage of alkylamines to the respective alkanals in Pseudomonas strain BERT is mediated by a PMS-dependent alkylamine dehydrogenase. This alkylamine dehydrogenase produces stoichiometric amounts of ammonium from octylamine. The PMS-dependent alkylamine was found to oxidize a broad range of long-chain alkylamines. PMS-dependent long-chain aldehyde dehydrogenase activity was also detected in cell-free extract of Pseudomonas strain BERT grown on octylamine. The proposed pathway for the oxidation of alkylamine in strain BERT proceeds from alkylamine to alkanal, and then to the fatty acid.     

Microbiological Studies of Coli-aerogenes Bacteria

α-Ketoglutarate was obtained in a very small amount by the oxidative fermentation of acetate with either a growing culture or the washed cells of Escherichia coli. This microorganism was also observed to accumulate a considerable amount of α-ketoglutarate as the oxidation-product of C₄-dicarboxylic acids such as succinate, fumarate, malate and oxalacetate. The addition of acetate to the reaction mixtures containing either C₃- or C₄-acids brought about an increase in the yield of α-ketoglutarate. The bacteria of coli-aerogenes revealed an ability of oxidizing tricarboxylic acids under suitable conditions, but there was no noticeable production of α-ketoglutarate. The formation of glyoxylate was observed to occur during the degradation of citrate by the bacteria of coli-aerogenes. Finally, a cyclic mechanism of aerobic carbon-metabolism in the bacteria was propounded and discussed.     

Phylogenetic Analysis of Dichloromethane-Utilizing Aerobic Methylotrophic Bacteria

The phylogenetic relationships of 12 aerobic dichloromethane-degrading bacteria that implement different C₁-assimilation pathways was determined based on 16S ribosomal RNA sequences and DNA–DNA hybridization data. The restricted facultative methylotroph Methylophilus leisingerii DM11 with the ribulose monophosphate pathway was found to belong to the genus Methylophilus cluster of the beta subclass of Proteobacteria. The facultative methylotroph Methylorhabdus multivorans DM13 was assigned to a separate branch of the alpha-2 group of Proteobacteria. Paracoccus methylutens DM12, which utilizes C₁-compounds via the Calvin cycle, was found to belong to the alpha-3 group of Proteobacteria (more precisely, to the genus Paracoccus cluster). Thus, phylogenetic analysis confirmed the taxonomic status of these recently characterized bacteria. According to the degree of DNA homology, several novel strains of methylotrophic bacteria were divided into three genotypic groups within the alpha-2 group of the Proteobacteria. Genotypic group 1, comprising strains DM1, DM3, and DM5 through DM9, and genotypic group 3, comprising strain DM10, were phylogenetically close to the methylotrophic bacteria of the genus Methylopila, whereas genotypic group 2 (strain DM4) was close to bacteria of the genus Methylobacterium. The genotypic groups obviously represent distinct taxa of methylotrophic bacteria, whose status should be confirmed by phenotypic analysis.     

Methylovorus mays sp. nov.: A new species of aerobic, obligately methylotrophic bacteria associated with plants

A bacterial strain utilizing methanol as the sole source of carbon and energy was isolated from the maize phyllosphere. Cells are nonpigmented gram-negative motile rods that do not form spores or prosthecae and reproduce by binary fission. The strain does not require vitamins or supplementary growth factors. It is obligately aerobic and urease-, oxidase-, and catalase-positive. The optimum growth temperature is 35–40°C; the optimum pH is 7.0–7.5. The doubling time is 2 h. The bacterium implements the ribulose monophosphate pathway and possesses NAD⁺-dependent 6-phosphogluconate dehydrogenase and enzymes of the glutamate cycle. α-Ketoglutarate dehydrogenase and enzymes of the glyoxylate cycle (isocitrate lyase and malate synthase) are absent. Fatty acids are dominated by palmitic (C_16:0) and palmitoleic (C_16:1) acids. The major phospholipids are phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine. Cardiolipin is present in minor amounts. The dominant ubiquinone is Q₈ The bacterial genome contains genes controlling the synthesis and secretion of cytokinins. The G+C content of DNA is 57.2 mol %, as determined from the DNA thermal denaturation temperature T_m. The bacterium shows low DNA homology (<10%) with restricted facultative methylotrophic bacteria of the genusMethylophilus (M. methylotrophus NCIMB 10515^T andM. leisingerii VKM B-2013¹) and with the obligate methylotrophic bacterium (Methylobacillus glycogenes ATCC 29475^T). DNA homology with the type representative of the genusMethylovorus, M. glucosetrophus VKM B-1745^T, is high (58%). The new isolate was classified as a new species,Methylovorus mays sp. nov.     

Bacterial degradation of glycol ethers

Assimilation of ethyleneglycol (EG) ethers by polyethyleneglycol-utilizing bacteria was examined. Ethyleneglycol ether-utilizing bacteria were also isolated from soil and activated sludge samples by enrichment-culture techniques. Three strains (4-5-3, EC 1-2-1 and MC 2-2-1) were selected and characterized as Pseudomonas sp. 4-5-3, Xanthobacter autotrophicus, and an unidentified gram-negative, non-spore-forming rod respectively. Their growth characteristics were examined: Pseudomonas sp. 4-5-3 assimilated EG (diethyleneglycol, DEG) monomethyl, monoethyl and monobutyl ethers, DEG, propanol and butanol. X. autotrophicus EC 1-2-1 grew well on EG monoethyl and monobutyl ethers, EG and primary alcohols (C₁-C₄), and slightly on EG monomethyl ether. The strain MC 2-2-1 grew on EG monomethyl ether, EG, primary alcohols (C₁-C₄), and 1,2-propyleneglycol (PG). The mixed culture of Pseudomonas sp. 4-5-3 and X. autotrophicus EC 1-2-1 showed better growth and improved degradation than respective single cultures towards EG monomethyl, monoethyl or monobutyl ethers. Intact cells of Pseudomonas sp. 4-5-3 degraded various kinds of monoalkyl ethers, which cannot be assimilated by the strain. Metabolic products were characterized from reaction supernatants of intact cells of Pseudomonas sp. 4-5-3 with EG or DEG monoethyl ethers: they were analyzed by thin-layer chromatography and GC-MS and found to be ethoxyacetic acid and ethoxyglycoxyacetic acid. Also, PG monoalkyl ethers (C₁-C₄), dipropyleneglycol monoethyl and monomethyl ethers and tripropyleneglycol monomethyl ether were assimilated by polypropyleneglycol-utilizing Corynebacterium sp. 7.     

The Apparent Malate Synthase Activity of Rhodobacter sphaeroides Is Due to Two Paralogous Enzymes, (3S)-Malyl-Coenzyme A (CoA)/β-Methylmalyl-CoA Lyase and (3S)- Malyl-CoA Thioesterase

Assimilation of acetyl coenzyme A (acetyl-CoA) is an essential process in many bacteria that proceeds via the glyoxylate cycle or the ethylmalonyl-CoA pathway. In both assimilation strategies, one of the final products is malate that is formed by the condensation of acetyl-CoA with glyoxylate. In the glyoxylate cycle this reaction is catalyzed by malate synthase, whereas in the ethylmalonyl-CoA pathway the reaction is separated into two proteins: malyl-CoA lyase, a well-known enzyme catalyzing the Claisen condensation of acetyl-CoA with glyoxylate and yielding malyl-CoA, and an unidentified malyl-CoA thioesterase that hydrolyzes malyl-CoA into malate and CoA. In this study the roles of Mcl1 and Mcl2, two malyl-CoA lyase homologs in Rhodobacter sphaeroides, were investigated by gene inactivation and biochemical studies. Mcl1 is a true (3S)-malyl-CoA lyase operating in the ethylmalonyl-CoA pathway. Notably, Mcl1 is a promiscuous enzyme and catalyzes not only the condensation of acetyl-CoA and glyoxylate but also the cleavage of β-methylmalyl-CoA into glyoxylate and propionyl-CoA during acetyl-CoA assimilation. In contrast, Mcl2 was shown to be the sought (3S)-malyl-CoA thioesterase in the ethylmalonyl-CoA pathway, which specifically hydrolyzes (3S)-malyl-CoA but does not use β-methylmalyl-CoA or catalyze a lyase or condensation reaction. The identification of Mcl2 as thioesterase extends the enzyme functions of malyl-CoA lyase homologs that have been known only as “Claisen condensation” enzymes so far. Mcl1 and Mcl2 are both related to malate synthase, an enzyme which catalyzes both a Claisen condensation and thioester hydrolysis reaction.Many organic compounds are initially metabolized to acetyl coenzyme A (acetyl-CoA), at which point they enter the central carbon metabolism. Examples of such growth substrates are C₁ and C₂ compounds (e.g., methanol and ethanol), fatty acids, waxes, esters, alkenes, or (poly)hydroxyalkanoates. The synthesis of all cell constituents from acetyl-CoA requires a specialized pathway for the conversion of this central C₂ unit into other biosynthetic precursor metabolites. This (anaplerotic) process is referred to as acetyl-CoA assimilation, and two very different strategies have been described, i.e., the glyoxylate cycle and the ethylmalonyl-CoA pathway (12, 21) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Pathways for acetyl-CoA assimilation. (A) Glyoxylate cycle. The key enzymes are isocitrate lyase and malate synthase. (B) Ethylmalonyl-CoA pathway. The unique enzymes of the pathway are crotonyl-CoA carboxylase/reductase, ethylmalonyl-CoA/methylmalonyl-CoA epimerase, (2R)-ethylmalonyl-CoA mutase, (2S)-methylsuccinyl-CoA dehydrogenase, mesaconyl-CoA hydratase, (3S)-malyl-CoA/β-methylmalonyl-CoA lyase, and (3S)-malyl-CoA thioesterase. The enzymes involved in the (apparent) malate synthase reaction(s) are boxed for each pathway.The glyoxylate cycle for acetyl-CoA assimilation is in fact a modified citric acid cycle that converts two molecules of acetyl-CoA to the citric acid cycle intermediate malate (Fig. ​(Fig.1A)1A) (21). In a first reaction sequence, one molecule of acetyl-CoA is converted into glyoxylate due to the combined action of the initial enzymes of the citric acid cycle and isocitrate lyase, the key enzyme of this assimilation strategy. Isocitrate lyase cleaves the citric cycle intermediate isocitrate into succinate and glyoxylate (22). The glyoxylate formed is then condensed in a second step with another molecule of acetyl-CoA to yield malate and free CoA. Because the two decarboxylation reactions of the citric acid cycle are circumvented by this acetyl-CoA assimilation strategy, the glyoxylate cycle is also referred to as the “glyoxylate bypass” or “glyoxylate shunt.”The ethylmalonyl-CoA pathway for acetyl-CoA assimilation replaces the glyoxylate cycle in bacteria that lack isocitrate lyase (1, 12). In this linear pathway, three molecules of acetyl-CoA, one molecule of CO₂, and one molecule of bicarbonate are converted to the citric acid cycle intermediates succinyl-CoA and malate (Fig. ​(Fig.1B).1B). The ethylmalonyl-CoA pathway requires at least seven unique enzymes. Crotonyl-CoA carboxylase/reductase, ethylmalonyl-CoA mutase, and methylsuccinyl-CoA dehydrogenase are considered key enzymes of the pathway, and all three enzymes have been characterized from Rhodobacter sphaeroides (12-14).Although these two acetyl-CoA strategies differ with respect to their reaction sequence, intermediates and overall balance, the glyoxylate cycle and the ethylmalonyl-CoA pathway both require the condensation of acetyl-CoA and glyoxylate to form malate (Fig. ​(Fig.1,1, boxed). In the glyoxylate cycle, this reaction is catalyzed by malate synthase, whereas in the ethylmalonyl-CoA pathway malate synthase is catalyzed by two separate enzymes, malyl-CoA lyase and malyl-CoA thioesterase (7, 26).Malyl-CoA lyases catalyze the reversible condensation of acetyl-CoA and glyoxylate into malyl-CoA and have been purified from Methylobacterium extorquens, Chloroflexus aurantiacus, Aminobacter aminovorans, and Rhodobacter capsulatus; the corresponding genes were identified as mclA (M. extorquens), mcl (C. aurantiacus), and mcl1 (R. capsulatus) (5, 16, 17, 19, 26). Remarkably, these proteins are promiscuous enzymes that also catalyze the (reversible) cleavage of β-methylmalyl-CoA into glyoxylate and propionyl-CoA, and it has been suggested that these enzymes catalyze both reactions in vivo (16, 19, 26). However, in contrast to malyl-CoA lyase, the malyl-CoA thioesterase catalyzing the highly exergonic hydrolysis of the CoA-thioester into malate and free CoA has not been identified so far, and the nature of the enzyme has remained enigmatic (7, 26).For R. sphaeroides, a malyl-CoA lyase homolog has been shown to be upregulated during growth on acetate, and it was proposed that this protein (Mcl1) catalyzes the cleavage of β-methylmalyl-CoA, as well as the condensation of acetyl-CoA and glyoxylate in the ethylmalonyl-CoA pathway (1). Interestingly, R. sphaeroides encodes a second malyl-CoA lyase homolog with 34% amino acid sequence identity to Mcl1. This protein, named Mcl2, was also shown to be upregulated during growth of R. sphaeroides on acetate, but a function could not be assigned so far (1). We therefore addressed the function of both malyl-CoA lyase homologs by gene inactivation and biochemical studies of recombinant Mcl1 and Mcl2. Based on our findings, we confirm here the function of Mcl1 in R. sphaeroides as (3S)-malyl-CoA/β-methylmalyl-CoA lyase and identify its paralog Mcl2 as the long-sought (3S)-malyl-CoA thioesterase.     

A reverse glyoxylate shunt to build a non-native route from C4 to C2 in Escherichia coli

Most central metabolic pathways such as glycolysis, fatty acid synthesis, and the TCA cycle have complementary pathways that run in the reverse direction to allow flexible storage and utilization of resources. However, the glyoxylate shunt, which allows for the synthesis of four-carbon TCA cycle intermediates from acetyl-CoA, has not been found to be reversible to date. As a result, glucose can only be converted to acetyl-CoA via the decarboxylation of the three-carbon molecule pyruvate in heterotrophs. A reverse glyoxylate shunt (rGS) could be extended into a pathway that converts C₄ carboxylates into two molecules of acetyl-CoA without loss of CO₂. Here, as a proof of concept, we engineered in Escherichia coli such a pathway to convert malate and succinate to oxaloacetate and two molecules of acetyl-CoA. We introduced ATP-coupled heterologous enzymes at the thermodynamically unfavorable steps to drive the pathway in the desired direction. This synthetic pathway in essence reverses the glyoxylate shunt at the expense of ATP. When integrated with central metabolism, this pathway has the potential to increase the carbon yield of acetate and biofuels from many carbon sources in heterotrophic microorganisms, and could be the basis of novel carbon fixation cycles.     

Compensatory effects of elevated CO<Subscript>2</Subscript> concentration on the inhibitory effects of high temperature and irradiance on photosynthetic gas exchange in carrots

We determined the interactive effects of irradiance, elevated CO₂ concentration (EC), and temperature in carrot (Daucus carota var. sativus). Plants of the cv. Red Core Chantenay (RCC) were grown in a controlled environmental plant growth room and exposed to 3 levels of photosynthetically active radiation (PAR) (400, 800, 1 200 μmol m⁻² s⁻¹), 3 leaf chamber temperatures (15, 20, 30 °C), and 2 external CO₂ concentrations (C _a), AC and EC (350 and 750 μmol mol⁻¹, respectively). Rates of net photosynthesis (P _N) and transpiration (E) and stomatal conductance (g _s ) were measured, along with water use efficiency (WUE) and ratio of internal and external CO₂ concentrations (C _i/C _a). P _N revealed an interactive effect between PAR and C _a. As PAR increased so did P _N under both C _a regimes. The g _s showed no interactive effects between the three parameters but had singular effects of temperature and PAR. E was strongly influenced by the combination of PAR and temperature. WUE was interactively affected by all three parameters. Maximum WUE occurred at 15 °C and 1 200 μmol m⁻² s^{− 1} PAR under EC. The C _i /C _a was influenced independently by temperature and C _a. Hence photosynthetic responses are interactively affected by changes in irradiance, external CO₂ concentration, and temperature. EC significantly compensates the inhibitory effects of high temperature and irradiance on P _N and WUE.     

Reactivity of glyoxylate with hydrogen perioxide and simulation of the glycolate pathway of C3 plants and Euglena

The nonenzymatic reaction of glyoxylate and H₂O₂ was measured under physiological conditions of the pH and concentrations of reactants. The reaction of glyoxylate and H₂O₂ was secondorder, with a rate constant of 2.27 l mol^-1 s^-1 at pH 8.0 and 25° C. The rate constant increased by 4.4 times in the presence of Zn²⁺ and doubled at 35°C. We propose a mechanism for the reaction between glyoxylate and H₂O₂. From a comparison of the rates of H₂O₂ decomposition by catalase and the reaction with glyoxylate, we conclude that H₂O₂ produced during glycolate oxidation in peroxisomes is decomposed by catalase but not by the reaction with glyoxylate, and that photorespiratory CO₂ originates from glycine, but not from glyoxylate, in C₃ plants. Simulation using the above rate constant and reported kinetic parameters leads to the same conclusion, and also makes it clear that alanine is a satisfactory amino donor in the conversion of glyoxylate to glycine. Some serine might be decomposed to give glycine and methylene-tetrahydrofolate; the latter is ultimately oxidized to CO₂. In the simulation of the glycolate pathway of Euglena, the rate constant was high enough to ensure the decarboxylation of glyoxylate by H₂O₂ to produce photorespiratory CO₂ during the glycolate metabolism of this organism.Abbreviations Chl chlorophyll - GGT glutamate: glyoxylate aminotransferase (EC 2.6.1.4) - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - SGT serine: glyoxylate aminotransferase (EC 2.6.1.45) This is the ninth in a series on the metabolism of glycolate in Euglena gracilis. The eighth is Yokota et al. (1982)     

Methylamine metabolism in a pseudomonas species

The mechanism by which a nonphotosynthetic bacterium Pseudomonas sp. (Shaw Strain MA) grows on the one-carbon source, methylamine, was investigated by comparing enzyme levels of cells grown on methylamine, to cells grown on acetate or succinate. Cells grown on methylamine have elevated levels of the enzymes serine hydroxymethyl transferase, serine dehydratase, malic enzyme, glycerate dehydrogenase and malate lyase (CoA acetylating ATP-cleaving). These enzymes, in conjunction with a constitutive glyoxylate transaminase, can account for the net conversion of two one-carbon units into acetyl CoA. Cells grown on acetate or methylamine, but not succinate, contain the enzyme isocitrate lyase; while cells grown on acetate or succinate, but not methylamine, contain significant levels of malate synthetase. These findings suggest that the acetyl CoA derived from one-carbon units in methylamine grown cells, condenses with oxalacetate to yield citrate and then isocitrate, followed by cleavage to succinate and glyoxylate. Thus, growth on methylamine is accomplished by the net synthesis of succinate from two molecules of methyamine and two molecules of CO₂.