Regulation of methanol oxidation and carbon dioxide fixation in Xanthobacter strain 25a grown in continuous culture

The regulation of C₁-metabolism in Xanthobacter strain 25a was studied during growth of the organism on acetate, formate and methanol in chemostat cultures. No activity of methanol dehydrogenase (MDH), formate dehydrogenase (FDS) or ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisC/O) could be detected in cells grown on acetate alone over a range of dilution rates tested. Addition of methanol or formate to the feed resulted in the immediate induction of MDH and FDH and complete utilization (D=0.10 h^-1) of acetate and the C ₁-substrates. The activities of these enzymes rapidly dropped at the higher growth rates, which suggests that their synthesis is further controlled via repression by heterotrophic substrates such as acetate. Synthesis of RuBisC/O already occurred at low methanol concentrations in the feed, resulting in additive growth yields on acetate/methanol mixtures. The energy generated in the oxidation of formate initially allowed an increased assimilation of acetate (and a decreased dissimilation), resulting in enhanced growth yields on the mixture. RuBisC/O activity could only be detected at the higher formate/acetate ratios in the feed. The data suggest that synthesis of RuBisC/O and CO₂ fixation via the Calvin cycle in Xanthobacter strain 25 a is controlled via a (de)repression mechanism, as is the case in other facultatively autotrophic bacteria. Autotrophic CO₂ fixation only occurs under conditions with a diminished supply of heterotrophic carbon sources and a sufficiently high availability of suitable energy sources. The latter point is further supported by the clearly more pronounced derepressing effect exerted by methanol compared to formate.Abbreviations FDH formate dehydrogenase - FBPase fructose-1,6-bisphosphatase - ICDH isocitrate dehydrogenase - MDH methanol dehydrogenase - PQQ pyrrolo quinoline quinone - PRK phosphoribulokinase - RuBisC/O ribulose-1,5-bisphosphate carboxylase/oxygenase - RuMP ribulose monophosphate - TCA tricarboxylic acid cycle     

Methanol and ethanol utilization in methylotrophic yeast Pichia pinus wild-type and mutant strains

In the wild-type strain of methylotrophic yeast Pichia pinus diauxic growth is observed during cultivation in medium containing a mixture of methanol and ethanol: firstly, slow phase of ethanol utilization is revealed and, secondly, a fast phase of methanol consumption is shown. Diauxic growth is observed also in ecr1 mutant, impaired in ethanol-induced catabolite repression of methylotrophic metabolism enzymes, but the order of utilization of the alcohols is inverted in this mutant. Such succession of alcohols utilization in both strains correlates well with the sequence of synthesis of microbody enzymes which catalyze key reactions of C₁- and C₂-metabolism. On the contrary, simultaneous utilization of methanol and ethanol from the mixture, as well as synchronous synthesis of both peroxisomal and glyoxisomal enzymes is observed in adh1 mutant which has reduced alcohol dehydrogenase activity. The strong differences between the wild-type strain and adh1 mutant were observed also in the kinetics of specific activity changes for C₁-metabolizing enzymes, localized in cytosol. In the wild-type strain during growth on methanol and ethanol mixture such changes correlate with the sequence of alcohol utilization. At the same time, in adh1 mutant the activities of formaldehyde dehydrogenase and formate dehydrogenase during the growth on the alcohols mixture are as high as during growth on methanol only, but the activity of dihydroxyacetone kinase is as low as under the growth on ethanol and is lower than on methanol.     

Toxicity of Mercaptoethanol to Mutant Strains of the Yeast Pichia methanolica Growing on Different Carbon Sources

Addition of -mercaptoethanol at a concentration of 2–3 mM to media containing methanol, glucose, or yeast extract caused a 50% inhibition of the growth of wild-type yeastPichia methanolica; mercaptoethanol at a concentration of 0.7 to 25 mM inhibited the growth of the mutant strain ecr1. The mutation mth1 of P. methanolica repressed its ability to consume methanol and was accompanied by the loss of alcohol oxidase (EC 1.1.3.13) activity. -Mercaptoethanol restored the ability of mth1 mutant cells to grow on methanol and stimulated their growth under derepression conditions. The growth effect of -mercaptoethanol during derepression was accompanied by partial restoration of alcohol oxidase activity.     

2-Bromoethanesulfonate: A selective agent for isolating resistantMethanosarcina mutants

Sodium 2-bromoethanesulfonate (BES), a structural analog of 2-mercaptoethanesulfonate (coenzyme M), inhibited methanogenesis and growth ofMethanosarcina strain 227 in the presence of H₂/CO₂, methanol, or acetate. A single exposure to 24 M BES was sufficient to produce cultures resistant to 240 M BES. Wild-type cultures inhibited by 200 M BES (or less) resumed growth and methane production when coenzyme M (coM) was added to the culture medium. Cultures incubated one week or longer with 200 M BES (or less) spontaneously resumed growth and methanogenesis in the presence of H₂/CO₂, methanol, or acetate without added coM. BES resistance was heritable and not the result of inactivation or decomposition of BES. BES resistance acquired on one methanogenic substrate was retained when cells were grown on a different methanogenic substrate. However, BES resistance did not confer multiple resistance to other halomethane compounds such as chloroform, 2-bromoethanol, 2-bromopropionic acid, and chloramphenicol. BES resistance varied in two other genera of methanogens tested. One strain ofMethanospirillum hungatei was very sensitive to BES, and no resistant mutants were demonstrated. One strain ofMethanobacterium formicicum, however, was resistant to 200 M BES without any known prior exposure to BES.     

The use of various substrates and substrate concentrations by a Hyphomicrobium sp. isolated from soil: Effect on growth rate and growth yield

The content of Hyphomicrobium sp. was estimated from a clay loam soil using the most probable number technique with methanol as the sole carbon source. The method enumerated Hyphomicrobia as 0.2% of the total bacteria determined by acridine orange direct counts. Hyphomicrobium sp. was not able to use C-C compounds such as glucose or acetate for growth. Maximal growth yield and growth rate were obtained when the concentration of methanol was in the range of 0.5–5 mg C/liter. Substrate affinity measurements revealed K_s values of 0.8 m and 5.8 m when the methanol concentration was 0.5–2.5 m and 5–200 m, respectively. Hyphomicrobium sp. had the ability to assimilate volatile organic compounds from air for growth. A growth yield of 0.7 mg/liter cell carbon was obtained in a mineral medium that contained no additions of organic compounds but had been stored for 4 weeks in flasks, allowing volatile compounds from the air to dissolve in the medium. When air was pumped into the culture during cultivating, the growth yield was proportional to the flow rate of air into the culture. Correspondence to: Kari Aa     

Influence of the counter anion and solvent in the structure of copper derivatives with the 2,3-bis(2-pyridyl)pyrazine ligand

Several compounds have been isolated from the reaction between different copper bis(acetylacetonato) derivatives and the potentially bridging ligand 2,3-bis(2-pyridyl)pyrazine (bppz). A compound of formula [Cu(tfacac)₂(bppz)] (1) is obtained when the substituted trifluoromethylacetylacetonato is used. The use of different anions and the unsubstituted acetylacetonato give rise to new derivatives of general formula [{Cu(acac)}₂(μ-bppz)₂]X₂ (X⁻ = BF₄⁻, 2; PF₆⁻, 3; BPh₄⁻, 4). In these compounds the bppz ligand is acting as a bridge by chelating one copper atom and bonding monodentate a second copper atom. The presence of anions with different coordination abilities introduces variations in the copper environment and geometry. When the non-coordinating tetraphenylborate is used different compounds depending on the nature of the solvent are obtained. The dimer 4 was isolated from a methanol/chloroform mixture, while in the absence of chloroform the monomeric compound of formula [Cu(acac)(bppz)(ROH)](BPh₄)·ROH (ROH = MeOH, 5) was obtained. When ethanol was used instead of methanol the analogous derivative 6 (R = EtOH) was isolated. Both species show a mononuclear structure with the copper atom five-coordinated by the chelating acac and bppz ligands and one hydroxo group occupying the apical position. A similar environment for the copper appears in [Cu(tfacac)(bppz)(MeOH)](BPh₄), 7, which shows a dimeric structure through hydrogen bonds interactions. The magnetic susceptibility data of the dimeric compounds show very weak antiferromagnetic interactions between the copper atoms, an expected fact since the bridging bppz ligand is not planar but the monodentate pyridine is more or less perpendicular to the other two aromatic rings, precluding the spin exchange via the π ligand electrons.     

Influence of sulphur-containing compounds on the growth of Methanosarcina barkeri in a defined medium

Summary Optimal growth of Methanosarcina barkeri occurred in a defined medium containing methanol when 2.5–4 mM sodium sulphide was added giving a concentration of 0.04–0.06 mM dissolved sulphide (HS^–+S^2–. When the sulphide concentration was too low for optimal growth (e.g., 0.1 mM Na₂S added) the addition of the redox resin Serdoxit acted as a sulphide reservoir and caused a significant stimulation of growth. Furthermore it could be demonstrated that iron sulphide, zinc sulphide or L-methionine could also act as sulphur sources while the addition of sodium sulphate to sulphide-depleted media failed to restore growth. The amino acid L-cysteine (0.85 mM) stimulated growth but could not replace Na₂S.Under optimal cysteine-and sulphide concentrations the generation time of this strain was about 7–9 h during growth on methanol, giving a growth yield of about 0.14 g/g methanol consumed. Different M. barkeri strains were also able to grow under these conditions on acetate (30–50 h doubling time) without a significant lag-phase and with complete substrate consumption even though the inoculum was grown on methanol or H₂–CO₂. When methanol and acetate were present as a mixture in the medium both were used simultaneously.     

Production of polyhydroxyalkanoates from methanol by a new methylotrophic bacterium Methylobacterium sp. GW2

A new bacterial strain, isolated from groundwater contaminated with explosives, was characterized as a pink-pigmented facultative methylotroph, affiliated to the genus Methylobacterium. The bacterial isolate designated as strain GW2 was found capable of producing the homopolymer poly-3-hydroxybutyrate (PHB) from various carbon sources such as methanol, ethanol, and succinate. Methanol acted as the best substrate for the production of PHB reaching 40 % w/w dry biomass. PHB accumulation was observed to be a growth-associated process, so that there was no need for two-step fermentation. Optimal growth occurred at 0.5 % (v/v) methanol concentration, and growth was strongly inhibited at concentration above 2 % (v/v). Methylobacterium sp. strain GW2 was also able to accumulate the copolyester poly-3-hydroxybutyrate-poly-3-hydroxyvalerate (PHB/HV) when valeric acid was supplied as an auxiliary carbon source to methanol. After 66 h, a copolymer content of 30 % (w/w) was achieved with a PHB to PHV ratio of 1:2. Biopolymers produced by strain GW2 had an average molecular weight ranging from 229,350 to 233,050 Da for homopolymer PHB and from 362,430 to 411,300 Da for the copolymer PHB/HV.     

Enhanced protein production by sorbitol co-feeding with methanol in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis> strains

Pichia pastoris strains carrying 1, 6, 12, and 18 copies of the porcine insulin precursor (PIP) gene, were employed to investigate the effects of sorbitol co-feeding with methanol on the physiology of the strains. Multicopy clones of the methylotrophic yeast were generated to vary the PIP gene dosage and recombinant proteins. Elevated gene dosage increased levels of the recombinant PIP protein when methanol served as the sole carbon and energy source i.e., an increase of 1.9% for a strain carrying 1 copy, 42.6% for a strain carrying 6 copies, 34.7% for a strain carrying 12 copies and 80.9% for a strain carrying 18 copies, respectively (using sorbitol co-feeding with methanol during the induction phase). However, it had no significant influence on a lower gene dosage strain (1 copy), but this approach affirmed enhancement in cell growth and PIP production for higher gene dosage strain (6, 12, and 18 copies) via using sorbitol co-feeding with methanol. Additionally, the co-feeding strategy could hold vital importance for recombinant protein production by a multi-copy P. pastoris system.     

Genomics and Biochemistry of Metabolic Pathways for the C<Subscript>1</Subscript> Compounds Utilization in Colorless Sulfur Bacterium <Emphasis Type="Italic">Beggiatoa leptomitoformis</Emphasis> D-402

The metabolic pathways of one-carbon compounds utilized by colorless sulfur bacterium Beggiatoa leptomitoformis D-402 were revealed based on comprehensive analysis of its genomic organization, together with physiological, biochemical and molecular biological approaches. Strain D-402 was capable of aerobic methylotrophic growth with methanol as a sole source of carbon and energy and was not capable of methanotrophic growth because of the absence of genes of methane monooxygenases. It was established that methanol can be oxidized to CO₂ in three consecutive stages. On the first stage methanol was oxidized to formaldehyde by the two PQQ (pyrroloquinolinequinone)-dependent methanol dehydrogenases (MDH): XoxF and Mdh2. Formaldehyde was further oxidized to formate via the tetrahydromethanopterin (H₄MPT) pathway. And on the third stage formate was converted to CO₂ by NAD⁺-dependent formate dehydrogenase Fdh2. Finally, it was established that endogenous CO₂, formed as a result of methanol oxidation, was subsequently assimilated for anabolism through the Calvin–Benson–Bassham cycle. The similar way of one-carbon compounds utilization also exists in representatives of another freshwater Beggiatoa species—B. alba.     

Enhanced truncated-t-PA (CT-b) expression in high-cell-density fed-batch cultures of <Emphasis Type="Italic">Pichia pastoris</Emphasis> through optimization of a mixed feeding strategy by response surface methodology

Recently, Pichia pastoris has been the focal point of interest as an expression system for production of many recombinant proteins. The study and optimization of feeding strategy are of major importance to achieve maximum volumetric productivity in fed-batch cultivations. Among different feeding strategies used in P. pastoris fed-batch cultures, those trying to maintain a constant specific growth rate have usually resulted in superior productivities. The objective of the present study was to investigate and optimize the co-feeding of glycerol and methanol to attain maximum expression of t-PA in P. pastoris fed-batch cultures with constant specific growth rate. The experiments were designed by response surface methodology, considering the specific feeding rates of methanol and glycerol as independent variables. In each experiment, glycerol and methanol were fed according to a predetermined equation to maintain a constant specific growth rate. It was found that with glycerol feeding for higher specific growth rates, the inhibitory properties of glycerol are more pronounced, while the best expression level was achieved when the ratio of µ _set glycerol to that of methanol was around 1.67. In all specific growth rates tested, almost a similar ratio of the specific glycerol feeding rate to that of methanol led to the maximum protein production and activity. The statistical model predicted the optimal operating conditions for µ _set glycerol and that of methanol to be 0.05 and 0.03 h^?1, respectively. Applying the optimum strategy, maximum of 52 g/L biomass, 300 mg/L t-PA and 340,000 IU/mL enzyme activity were obtained.     

Saccharomyces cerevisiae wine strains differing in copper resistance exhibit different capability to reduce copper content in wine

Two wine strains of Saccharomyces cerevisiae, characterized by a different degree of copper resistance, were tested in grape must fermentation in the presence of different copper concentrations. The sensitive strain SN9 was strongly affected by copper concentration (32 ppm, (32 mg/l)), whereas the resistant strain SN41 exhibited a good growth activity in presence of 32 ppm of copper and only a reduced activity in presence of 320 ppm. The different strain fermentation performance in response to the copper addition corresponded to a different capability to accumulate copper inside the cells. Both strains exhibited the capacity to reduce the copper content in the final product, eventhough a significantly greater reducing activity was exerted by the resistant strain SN41, which was able to reduce by 90% the copper concentration in the final product and to accumulate the metal in great concentrations in the cells. As high concentrations of copper can be responsible for wine alterations, the selection of S. cerevisiae strains possessing high copper resistance and the ability to reduce the copper content of wine has a great technological interest, in particular for the fermentation of biological products. From the results obtained, the technique proposed is not only suitable for the assay of copper residues in must, wine and yeast cells, but it also offers the advantage of easy sample preparation and low detection limit in the ppb (g/l) range.     

The production of azurin and similar proteins

Summary It has been shown that, in the organisms tested, the production of azurin or similar blue, copper-protein complexes is confined to bacterial species of the three genera Bordetella, Alcaligenes and Pseudomonas. In the strain of Pseudomonas aeruginosa used, there appeared to be no difference in the amount of azurin occurring when the strain was grown aerobically and anaerobically. The amount of azurin produced by representative strains of Ps. aeruginosa, B. bronchiseptica and A. denitrificans varied with the copper content of the medium. Above a level of 5 g copper/ml of medium, the azurin content was constant for the three species tested; below a copper level of 0.5 g/ml there was an almost total absence of azurin although good growth occurred. Under similar growth conditions, the azurin content of the three bacterial species studied was not significantly different.The possible role of azurin is discussed.     

Herbicidal activity of the antibiotics geldanamycin and nigericin

Geldanamycin and nigericin, phytotoxic metabolites from a strain ofStreptomyces hygroscopicus, were tested for herbicidal activity and selectivity on a range of crop and weed species. In petri dish bioassays, geldanamycin reduced radicle growth of all species tested, whereas nigericin inhibited 7 of 10. The two compounds in mixture appeared to be additive rather than synergistic in effect. In assays with seeds and seedlings in field soil, geldanamycin showed significant preemergence activity on proso millet, barnyardgrass, garden cress, and giant foxtail. It had no postemergence herbicidal effect on any of the species tested. Nigericin had preemergence activity on garden cress and large crabgrass and postemergence activity on garden cress and velvetleaf. The postemergence effect of nigericin on velvetleaf was especially striking, with leaves showing symptoms of injury within 24–48 h of treatment. Doses as low as 0.3 kg/ha caused damage. The primary herbicidal effect of both compounds was slowing of seed germination or seedling growth, although some plants were killed, especially at higher rates of application. Herbicidal effects were most pronounced for 1 to 2 weeks after treatment and diminished thereafter.     

Isolation and characterization of an obligate methanol-utilizing bacteriumMethylomonas M-15

Summary A new obligate methylotrophic bacterium capable of rapid growth on methanol as its sole carbon and energy source was isolated. The organism grows only on methanol and not on methane or methylamine. It is a gram-negative, motile rod (0.5×1.5 m) with a single polar flagellum and was therefore classified as a species ofMethylomonas.The doubling time was about 2 hr at pH 7.0, at a temperature of 30 to 36°C and at a methanol concentration of 1% (v/v).Cell suspensions were able to oxidize methanol, formaldehyde, and formate; therefore it seems that methanol-, formaldehyde-, and formate-oxidizing enzymes are present in the bacterium. While no hydroxy pyruvate reductase activity was found, the cell extract contained high hexulose phosphate-synthetase activity, indicating the assimilation of methanol via the ribulose phosphate-pathway.The protein content of the bacterium is 60% and the amino acid pattern indicates that this strain could serve as a good source of single cell protein.     

Development of a bisphenol A-adsorbing yeast by surface display of the<Emphasis Type="Italic"> Kluyveromyces</Emphasis> yellow enzyme on<Emphasis Type="Italic"> Pichia pastoris</Emphasis>

A novel surface-engineered strain of yeast Pichia pastoris was constructed that displays at its surface Kluyveromyces lactis Yellow Enzyme (KYE) fused to the C-terminal half of Saccharomyces cerevisiae -agglutinin. The expression of the fusion protein was controlled by the AOX1-promoter. The new strain showed an increased sorption of the xenoestrogen Bisphenol A (BPA). It was shown that sorption of BPA depended on the presence of methanol in the growth medium and on the pH of the binding assays. The binding kinetics were typical for binding at a surface. The present results demonstrate that the -agglutinin surface display system can be used in the yeast P. pastoris.     

One carbon metabolism in methanogenic bacteria

Two strains of Methanosarcina (M. Barkeri strain MS, isolated from sewage sludge, and strain UBS, isolated from lake sediments) were found to have similar cellular properties and to have DNA base compositions of 44 mol percent guanosine plus cytosine. Strain MS was selected for further studies of its one-carbon metabolism. M. barkeri grew autotrophically via H₂ oxidation/CO₂ reduction. The optimum temperature for growth and methanogenesis was 37°C. H₂ oxidation proceeded via an F₄₂₀-dependent NADP⁺-linked hydrogenase. A maximum specific activity of hydrogenase in cell-free extracts, using methyl viologen as electron acceptor, was 6.0 mol min · mg protein at 37°C and the optimum pH (9.0). M. barkeri also fermented methanol andmethylamine as sole energy sources for growth. Cell yields during growth on H₂/CO₂ and on methanol were 6.4 and 7.2 mg cell dry weight per mmol CH₄ formed, respectively. During mixotrophic growth on H₂/CO₂ plus methanol, most methane was derived from methanol rather than from CO₂. Similar activities of hydrogenase were observed in cell-free extracts from H₂/CO₂-grown and methanol-grown cells. Methanol oxidation apparently proceeded via carrierbound intermediates, as no methylotrophy-type of methanol dehydrogenase activity was observed in cell-free extracts. During growth on methanol/CO₂, up to 48% of the cell carbon was derived from methanol indicating that equivalent amounts of cell carbon were derived from CO₂ and from an organic intermediate more reduced than CO₂. Cell-free extracts lacked activity for key cell carbon synthesis enzymes of the Calvin cycle, serine path, or hexulose path.Abbreviations CAPS cycloaminopropane sulfonic acid - CH₃-SCoM methyl coenzyme M - DCPIP 2,6-dichlorophenolindophenol - DEAE diethylaminoethyl - dimethyl POPOP 1,4-bis-2-(4-mothyl-5-phenyloxazolyl)-benzene - DNA deoxyribonucleic acid - dpm dismtegrations per min - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - F₄₂₀ factor 420 - G+C guanosine plus cytosine - NAD⁺ nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - PBBW phosphate buffered basal Weimer - PMS phenazine methosulfate - PPO 2,5-diphenyloxazole - rRNA ribosomal ribonucleic acid - RuBP ribulose-1,5-bisphosphate - Tris tris-hydroxymethyl-aminomethane - max maximum specific growth rate     

The persistence of bioluminescent Rhizobium meliloti strains L1 (RecA-) and L33 (RecA+) in non-sterile microcosms depends on the soil type,on the co-cultivation of the host legume alfalfa and on the presence of an indigenous R. meliloti population

In comparative analyses the influence of soil type, the rhizosphere of plants and the presence of an indigenous R. meliloti population on the population dynamics of bioluminescent R. meliloti strains L1 (RecA-) and L33 (RecA+) was assessed in microcosm studies. Both strains established better in a loamy and a clayey soil compared to a sandy soil. RecA- strain L1 showed a slightly but statistically significant reduced survival ability compared to RecA+ strain L33 (p 0.05). The presence of the host plant alfalfa stimulated the growth of both strains in non-sterile soil and no differences in the survival between both strains were observed. Co-cultivation of clover or wheat plants, respectively, neither positively nor negatively influenced the strains' survival. The most pronounced effect on the survival of both strains was exerted by the presence of an indigenous R. meliloti population. RecA- strain L1 showed a significantly impaired survival compared to RecA+ strain L33 (p 0.002). Moreover, no growth stimulation of strains L1 and L33 by the presence of the host plant alfalfa could be observed. These results indicate that the recA mutation affects the long-term rather than the short-term persistence of R. meliloti after environmental release.     

Metabolomics Revealed an Association of Metabolite Changes and Defective Growth in Methylobacterium extorquens AM1 Overexpressing ecm during Growth on Methanol

Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and multi-carbon compounds. The ethylmalonyl-CoA (EMC) pathway is one of the central assimilatory pathways in M. extorquens during growth on C1 and C2 substrates. Previous studies had shown that ethylmalonyl-CoA mutase functioned as a control point during the transition from growth on succinate to growth on ethylamine. In this study we overexpressed ecm, phaA, mcmAB and found that upregulating ecm by expressing it from the strong constitutive mxaF promoter caused a 27% decrease in growth rate on methanol compared to the strain with an empty vector. Targeted metabolomics demonstrated that most of the central intermediates in the ecm over-expressing strain did not change significantly compared to the control strain; However, poly-β-hydroxybutyrate (PHB) was 4.5-fold lower and 3-hydroxybutyryl-CoA was 1.6-fold higher. Moreover, glyoxylate, a toxic and highly regulated essential intermediate, was determined to be 2.6-fold higher when ecm was overexpressed. These results demonstrated that overexpressing ecm can manipulate carbon flux through the EMC pathway and divert it from the carbon and energy storage product PHB, leading to an accumulation of glyoxylate. Furthermore, untargeted metabolomics discovered two unusual metabolites, alanine (Ala)–meso-diaminopimelic acid (mDAP) and Ala–mDAP–Ala, each over 45-fold higher in the ecm over-expressing strain. These two peptides were also found to be highly produced in a dose-dependent manner when glyoxylate was added to the control strain. Overall, this work has explained a direct association of ecm overexpression with glyoxylate accumulation up to a toxic level, which inhibits cell growth on methanol. This research provides useful insight for manipulating the EMC pathway for efficiently producing high-value chemicals in M. extorquens.