Ribulose Monophosphate Pathway Research Articles

Methylomarinovum tepidoasis sp. nov., a moderately thermophilic methanotroph of the family Methylothermaceae isolated from a deep-sea hydrothermal field.

A novel aerobic methanotrophic bacterium, designated as strain IN45T, was isolated from in situ colonisation systems deployed at the Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough. IN45T was a moderately thermophilic obligate methanotroph that grew only on methane or methanol at temperatures between 25 and 56 °C (optimum 45-50 °C). It was an oval-shaped, Gram-reaction-negative, motile bacterium with a single polar flagellum and an intracytoplasmic membrane system. It required 1.5-4.0 % (w/v) NaCl (optimum 2-3 %) for growth. The major phospholipid fatty acids were C16 : 1ω7c, C16 : 0 and C18 : 1ω7c. The major isoprenoid quinone was Q-8. The 16S rRNA gene sequence comparison revealed 99.1 % sequence identity with Methylomarinovum caldicuralii IT-9T, the only species of the genus Methylomarinovum with a validly published name within the family Methylothermaceae. The complete genome sequence of IN45T consisted of a 2.42-Mbp chromosome (DNA G+C content, 64.1 mol%) and a 20.5-kbp plasmid. The genome encodes genes for particulate methane monooxygenase and two types of methanol dehydrogenase (mxaFI and xoxF). Genes involved in the ribulose monophosphate pathway for carbon assimilation are encoded, but the transaldolase gene was not found. The genome indicated that IN45T performs partial denitrification of nitrate to N2O, and its occurrence was indirectly confirmed by N2O production in cultures grown with nitrate. Genomic relatedness indices between the complete genome sequences of IN45T and M. caldicuralii IT-9T, such as digital DNA-DNA hybridisation (51.2 %), average nucleotide identity (92.94 %) and average amino acid identity (93.21 %), indicated that these two methanotrophs should be separated at the species level. On the basis of these results, strain IN45T represents a novel species, for which we propose the name Methylomarinovum tepidoasis sp. nov. with IN45T (=JCM 35101T =DSM 113422T) as the type strain.

Methylomonadaceae was the active and dominant methanotroph in Tibet lake sediments.

Methane (CH4), an important greenhouse gas, significantly impacts the local and global climate. Our study focused on the composition and activity of methanotrophs residing in the lakes on the Tibetan Plateau, a hotspot for climate change research. Based on the field survey, the family Methylomonadaceae had a much higher relative abundance in freshwater lakes than in brackish and saline lakes, accounting for ~92% of total aerobic methanotrophs. Using the microcosm sediment incubation with 13CH4 followed by high throughput sequencing and metagenomic analysis, we further demonstrated that the family Methylomonadaceae was actively oxidizing CH4. Moreover, various methylotrophs, such as the genera Methylotenera and Methylophilus, were detected in the 13C-labeled DNAs, which suggested their participation in CH4-carbon sequential assimilation. The presence of CH4 metabolism, such as the tetrahydromethanopterin and the ribulose monophosphate pathways, was identified in the metagenome-assembled genomes of the family Methylomonadaceae. Furthermore, they had the potential to adapt to oxygen-deficient conditions and utilize multiple electron acceptors, such as metal oxides (Fe3+), nitrate, and nitrite, for survival in the Tibet lakes. Our findings highlighted the predominance of Methylomonadaceae and the associated microbes as active CH4 consumers, potentially regulating the CH4 emissions in the Tibet freshwater lakes. These insights contributed to understanding the plateau carbon cycle and emphasized the significance of methanotrophs in mitigating climate change.

Engineering Escherichia coli for Poly-β-hydroxybutyrate Production from Methanol.

The naturally occurring one-carbon assimilation pathways for the production of acetyl-CoA and its derivatives often have low product yields because of carbon loss as CO2. We constructed a methanol assimilation pathway to produce poly-3-hydroxybutyrate (P3HB) using the MCC pathway, which included the ribulose monophosphate (RuMP) pathway for methanol assimilation and non-oxidative glycolysis (NOG) for acetyl-CoA (precursor for PHB synthesis) production. The theoretical product carbon yield of the new pathway is 100%, hence no carbon loss. We constructed this pathway in E. coli JM109 by introducing methanol dehydrogenase (Mdh), a fused Hps-phi (hexulose-6-phosphate synthase and 3-phospho-6-hexuloisomerase), phosphoketolase, and the genes for PHB synthesis. We also knocked out the frmA gene (encoding formaldehyde dehydrogenase) to prevent the dehydrogenation of formaldehyde to formate. Mdh is the primary rate-limiting enzyme in methanol uptake; thus, we compared the activities of three Mdhs in vitro and in vivo and then selected the one from Bacillus methanolicus MGA3 for further study. Experimental results indicate that, in agreement with the computational analysis results, the introduction of the NOG pathway is essential for improving PHB production (65% increase in PHB concentration, up to 6.19% of dry cell weight). We demonstrated that PHB can be produced from methanol via metabolic engineering, which provides the foundation for the future large-scale use of one-carbon compounds for biopolymer production.

Engineering and adaptive laboratory evolution of Escherichia coli for improving methanol utilization based on a hybrid methanol assimilation pathway.

Engineering Escherichia coli for efficient methanol assimilation is important for developing methanol as an emerging next-generation feedstock for industrial biotechnology. While recent attempts to engineer E. coli as a synthetic methylotroph have achieved great success, most of these works are based on the engineering of the prokaryotic ribulose monophosphate (RuMP) pathway. In this study, we introduced a hybrid methanol assimilation pathway which consists of prokaryotic methanol dehydrogenase (Mdh) and eukaryotic xylulose monophosphate (XuMP) pathway enzyme dihydroxyacetone synthase (Das) into E. coli and reprogrammed E. coli metabolism to improve methanol assimilation by combining rational design and adaptive laboratory evolution. By deletion and down-regulation of key genes in the TCA cycle and glycolysis to increase the flux toward the cyclic XuMP pathway, methanol consumption and the assimilation of methanol to biomass were significantly improved. Further improvements in methanol utilization and cell growth were achieved via adaptive laboratory evolution and a final evolved strain can grow on methanol with only 0.1g/L yeast extract as co-substrate. 13C-methanol labeling assay demonstrated significantly higher labeling in intracellular metabolites in glycolysis, TCA cycle, pentose phosphate pathway, and amino acids. Transcriptomics analysis showed that the expression of fba, dhak, and part of pentose phosphate pathway genes were highly up-regulated, suggesting that the rational engineering strategies and adaptive evolution are effective for activating the cyclic XuMP pathway. This study demonstrated the feasibility and provided new strategies to construct synthetic methylotrophy of E. coli based on the hybrid methanol assimilation pathway with Mdh and Das.

Interactions among deep-sea mussels and their epibiotic and endosymbiotic chemoautotrophic bacteria: Insights from multi-omics analysis.

Endosymbiosis with Gammaproteobacteria is fundamental for the success of bathymodioline mussels in deep-sea chemosynthesis-based ecosystems. However, the recent discovery of Campylobacteria on the gill surfaces of these mussels suggests that these host-bacterial relationships may be more complex than previously thought. Using the cold-seep mussel ( Gigantidas haimaensis) as a model, we explored this host-bacterial system by assembling the host transcriptome and genomes of its epibiotic Campylobacteria and endosymbiotic Gammaproteobacteria and quantifying their gene and protein expression levels. We found that the epibiont applies a sulfur oxidizing (SOX) multienzyme complex with the acquisition of soxB from Gammaproteobacteria for energy production and switched from a reductive tricarboxylic acid (rTCA) cycle to a Calvin-Benson-Bassham (CBB) cycle for carbon assimilation. The host provides metabolic intermediates, inorganic carbon, and thiosulfate to satisfy the materials and energy requirements of the epibiont, but whether the epibiont benefits the host is unclear. The endosymbiont adopts methane oxidation and the ribulose monophosphate pathway (RuMP) for energy production, providing the major source of energy for itself and the host. The host obtains most of its nutrients, such as lysine, glutamine, valine, isoleucine, leucine, histidine, and folate, from the endosymbiont. In addition, host pattern recognition receptors, including toll-like receptors, peptidoglycan recognition proteins, and C-type lectins, may participate in bacterial infection, maintenance, and population regulation. Overall, this study provides insights into the complex host-bacterial relationships that have enabled mussels and bacteria to thrive in deep-sea chemosynthetic ecosystems.

Cytobacillus pseudoceanisediminis sp. nov., A Novel Facultative Methylotrophic Bacterium with High Heavy Metal Resistance Isolated from the Deep Underground Saline Spring

In this study, we present the characterization of the BNO1T bacterial strain isolated from the deep subsurface saline spring at the Baksan Neutrino Observatory INR RAS (Kabardino-Balkaria, Russia). The complete genome sequence of the strain BNO1T is 5,347,902bp, with a GC content 41 and 49%. The cell wall peptidoglycan contains meso-diaminopimelic acid. The major isoprenoid quinone is MK-7 and the polar lipids are diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The major fatty acids are anteiso-C15:0 (23.34%), iso-C15:0 (20.10%), C16:0 (11.96%), iso-C16:0 (10.88%), and anteiso-C17:0 (10.79%). The 16S rRNA gene sequence clearly demarcated the strain as belonging to Cytobacillus genera. Based on the phylogenetic analysis, ANI (average nucleotide identity) and dDDH (digital DNA-DNA hybridization) assessments we propose to assign the strain BNO1T and other related strains to new species and to name it Cytobacillus pseudoceanisediminis sp. nov. (The values of ANI and dDDH between BNO1T and Cytobacillus oceanisediminis CGMCC 1.10115T are 80.65% and 24.7%, respectively; values of ANI and dDDH between BNO1T and Cytobacillus firmus NCTC 10335T are 89% and 38%, respectively). Genomic analysis of strain BNO1T revealed pathways for C1 compounds oxidation and two pathways for C1 compounds assimilation: serine and ribulose monophosphate pathways. In addition, strain BNO1T contains a plasmid (342,541bp) coding multiple genes involved in heavy metal ion balance. Moreover, heavy metal toxicity testing confirmed the high potential of the strain BNO1T as a source of metal resistance genes and enzymes. The type strain is BNO1T (= BIM B-1921T = VKM B-3664T).

Methanotrophy by a Mycobacterium species that dominates a cave microbial ecosystem.

So far, only members of the bacterial phyla Proteobacteria and Verrucomicrobia are known to grow methanotrophically under aerobic conditions. Here we report that this metabolic trait is also observed within the Actinobacteria. We enriched and cultivated a methanotrophic Mycobacterium from an extremely acidic biofilm growing on a cave wall at a gaseous chemocline interface between volcanic gases and the Earth's atmosphere. This Mycobacterium, for which we propose the name Candidatus Mycobacterium methanotrophicum, is closely related to well-known obligate pathogens such as M. tuberculosis and M. leprae. Genomic and proteomic analyses revealed that Candidatus M. methanotrophicum expresses a full suite of enzymes required for aerobic growth on methane, including a soluble methane monooxygenase that catalyses the hydroxylation of methane to methanol and enzymes involved in formaldehyde fixation via the ribulose monophosphate pathway. Growth experiments combined with stable isotope probing using 13C-labelled methane confirmed that Candidatus M. methanotrophicum can grow on methane as a sole carbon and energy source. A broader survey based on 16S metabarcoding suggests that species closely related to Candidatus M. methanotrophicum may be abundant in low-pH, high-methane environments.

Connecting Algal Polysaccharide Degradation to Formaldehyde Detoxification.

Formaldehyde is a toxic metabolite that is formed in large quantities during bacterial utilization of the methoxy sugar 6‐O‐methyl‐d‐galactose, an abundant monosaccharide in the red algal polysaccharide porphyran. Marine bacteria capable of metabolizing porphyran must therefore possess suitable detoxification systems for formaldehyde. We demonstrate here that detoxification of formaldehyde in the marine Flavobacterium Zobellia galactanivorans proceeds via the ribulose monophosphate pathway. Simultaneously, we show that the genes encoding the key enzymes of this pathway are important for maintaining high formaldehyde resistance. Additionally, these genes are upregulated in the presence of porphyran, allowing us to connect porphyran degradation to the detoxification of formed formaldehyde.

Elucidation of dominant energy metabolic pathways of methane, sulphur and nitrogen in respect to mangrove-degradation for climate change mitigation

Mangroves play a key role in ecosystem balancing and climate change mitigation. It acts as a source and sink of methane (CH4), a major greenhouse gas responsible for climate change. Energy metabolic pathways of methane production (methanogenesis) and oxidation (methanotrophy) are directly driven by sulphur (S) and nitrogen (N) metabolism and salinity in coastal wetlands. To investigate, how mangrove-degradations, affect the source-sink behaviour of CH4; the pathways of CH4, S and N were studied through whole-genome metagenomic approach. Soil samples were collected from degraded and undisturbed mangrove systems in Sundarban, India. Structural and functional microbial diversities (KEGG pathways) of CH4, S and N metabolism were analysed and correlated with labile carbon pools and physico-chemical properties of soil. Overall, the acetoclastic pathway of methanogenesis was dominant. However, the relative proportion of conversion of CO2 to CH4 was more in degraded mangroves. Methane oxidation was higher in undisturbed mangroves and the serine pathway was dominant. After serine, the ribulose monophosphate pathway of CH4 oxidation was dominant in degraded mangrove, while the xylulose monophosphate pathway was dominant in undisturbed site as it is more tolerant to salinity and higher pH. The assimilatory pathway (AMP) of S-metabolism was dominant in both systems. But in AMP pathway, adenosine triphosphate sulfurylase enzyme reads were higher in degraded mangrove, while NADPH-sulfite reductase abundance was higher in undisturbed mangrove due to higher salinity, and pH. In N-metabolism, the denitrification pathway was predominant in degraded sites, whereas the dissimilatory nitrate reduction pathway was dominant in undisturbed mangroves. The relative ratios of sulphur reducing bacteria (SRB): methanogens were higher in degraded mangrove; however, methanotrophs:methanogens was higher in undisturbed mangrove indicated lower source and greater sink capacity of CH4 in the system. Microbial manipulation in mangrove-rhizosphere for regulating major energy metabolic pathways of methane could open-up a new window of climate change mitigation in coastal wetlands.

Carbon and hydrogen isotope fractionation during aerobic oxidation of short-chain alkanes in experimental incubations of vent fluids

Aerobic oxidation of short-chain alkanes was observed in gas samples from the Lutao intertidal hydrothermal vents in Taiwan, during storage at 20 °C for up to 29 months without adding bacterial strains and replenishing substrates. The carbon isotope fractionation factors (εC) of methane (C1), ethane (C2), and propane (C3), were calculated using the Rayleigh fractionation equation to be −37.1 ± 7.5‰, −14.8 ± 4.8‰, and −4.7 ± 5.2‰, respectively. The hydrogen isotope fractionation factor (εH) of methane was determined to be −281 ± 187‰. DNA sequencing of the 16S rRNA gene in the vent fluids suggests that aerobic oxidation is dominated by methanotrophs of the genera Methylomicrobium and Methylophaga, which use the ribulose monophosphate pathway (RuMP). The degrees of isotope fractionation (εC and εH values) herein are larger than previously reported values, possibly due to the limited O2 supply and low abundance of aerobic methane-oxidizing bacteria in the experiments. Since the fractionation factor of methane is higher than those of ethane and propane, the aerobic oxidation of thermogenic or microbial alkanes could produce a carbon isotope reversal, which is frequently noted as a trait of abiotic hydrocarbons. This work demonstrates that in addition to anaerobic microbial oxidation, aerobic oxidation with a low cell density can also produce significant isotope fractionation of alkanes in geological closed/semi-closed environments and open flow reaction systems that are characterized by moderate temperatures and a limited supply of substrates and O2; these environments include cold seeps, mud volcanoes, and low-temperature hydrothermal plumes/aquifers/reservoirs.

Metagenome Assembled Genome of a Novel Verrucomicrobial Methanotroph From Pantelleria Island.

Verrucomicrobial methanotrophs are a group of aerobic bacteria isolated from volcanic environments. They are acidophiles, characterized by the presence of a particulate methane monooxygenase (pMMO) and a XoxF-type methanol dehydrogenase (MDH). Metagenomic analysis of DNA extracted from the soil of Favara Grande, a geothermal area on Pantelleria Island, Italy, revealed the presence of two verrucomicrobial Metagenome Assembled Genomes (MAGs). One of these MAGs did not phylogenetically classify within any existing genus. After extensive analysis of the MAG, we propose the name of “Candidatus Methylacidithermus pantelleriae” PQ17 gen. nov. sp. nov. The MAG consisted of 2,466,655 bp, 71 contigs and 3,127 predicted coding sequences. Completeness was found at 98.6% and contamination at 1.3%. Genes encoding the pMMO and XoxF-MDH were identified. Inorganic carbon fixation might use the Calvin-Benson-Bassham cycle since all genes were identified. The serine and ribulose monophosphate pathways were incomplete. The detoxification of formaldehyde could follow the tetrahydrofolate pathway. Furthermore, “Ca. Methylacidithermus pantelleriae” might be capable of nitric oxide reduction but genes for dissimilatory nitrate reduction and nitrogen fixation were not identified. Unlike other verrucomicrobial methanotrophs, genes encoding for enzymes involved in hydrogen oxidation could not be found. In conclusion, the discovery of this new MAG expands the diversity and metabolism of verrucomicrobial methanotrophs.

Methanol-Essential Growth of Corynebacterium glutamicum: Adaptive Laboratory Evolution Overcomes Limitation due to Methanethiol Assimilation Pathway.

Methanol is a sustainable substrate for biotechnology. In addition to natural methylotrophs, metabolic engineering has gained attention for transfer of methylotrophy. Here, we engineered Corynebacterium glutamicum for methanol-dependent growth with a sugar co-substrate. Heterologous expression of genes for methanol dehydrogenase from Bacillus methanolicus and of ribulose monophosphate pathway genes for hexulose phosphate synthase and isomerase from Bacillus subtilis enabled methanol-dependent growth of mutants carrying one of two independent metabolic cut-offs, i.e., either lacking ribose-5-phosphate isomerase or ribulose-5-phosphate epimerase. Whole genome sequencing of strains selected by adaptive laboratory evolution (ALE) for faster methanol-dependent growth was performed. Subsequently, three mutations were identified that caused improved methanol-dependent growth by (1) increased plasmid copy numbers, (2) enhanced riboflavin supply and (3) reduced formation of the methionine-analogue O-methyl-homoserine in the methanethiol pathway. Our findings serve as a foundation for the engineering of C. glutamicum to unleash the full potential of methanol as a carbon source in biotechnological processes.

Methylotenera oryzisoli sp. nov., a lanthanide-dependent methylotrophic bacteria isolated from rice field soil.

A new lanthanide (Ln3+)-dependent methanol-utilizing bacterial strain, La3113T, was isolated from rice field soil and its taxonomic position was investigated using polyphasic approaches. The strain was aerobic, Gram-stain-negative, strongly motile, catalase-positive and cytochrome oxidase-positive. It could neither catalyse the hydrolysis of urea nor reduce nitrate to nitrite. Growth was observed within a temperature range of 10-40 °C and a pH range of 6-8, with optimum growth at 28 °C and pH 7. Methylamine was utilized as the single source of energy, carbon and nitrogen, and it was oxidized by methylamine dehydrogenase. C16 : 1 ω7c, C16 : 1 ω6c and C16 : 0 were the dominant cellular fatty acids. Its draft genome (2.67 Mbp and 44.9 mol% G+C content) encodes genes including three Ln3+-dependent methanol dehydrogenase (XoxF-type MDH) genes, those for formaldehyde assimilation (ribulose monophosphate pathway), formate dehydrogenases and methylamine dehydrogenases, but not Ca2+-dependent MDH (MxaFI-MDH), which characterizes the species as a Ln3+-dependent methylotroph. The 16S rRNA gene sequence showed that strain La3113T belongs to the genus Methylotenera and is closely related to Methylotenera mobilis JLW8T (98.29 % identity). The digital DNA-DNA hybridization (dDDH) values (less than 30 %) and average nucleotide identity (ANI) values (less than 85 %) between genomes of strain La3113T and related type strains were lower than the thresholds for species delineation (70 % for dDDH and 95-96 % for ANI). On the basis of these polyphasic approaches, we propose a novel Methylotenera species, Methylotenera oryzisoli sp. nov. (type strain La3113T=NBRC 111954T=DSM 103219T).

An Aldolase-Catalyzed New Metabolic Pathway for the Assimilation of Formaldehyde and Methanol To Synthesize 2-Keto-4-hydroxybutyrate and 1,3-Propanediol in Escherichia coli.

Formaldehyde (HCHO) is an important intermediate in the metabolism of one-carbon (C1) compounds such as methanol, formate, and methane. The ribulose monophosphate (RuMP) pathway is the most-studied HCHO assimilation route and the 3-hexulose-6-phosphate synthase (Hps) plays an important role for HCHO fixation. In this study, we proposed and selected a pyruvate-dependent aldolase to channel HCHO into 2-keto-4-hydroxybutyrate as an important intermediate for biosynthesis. By combining this reaction with three further enzymes we demonstrated a pyruvate-based C1 metabolic pathway for biosynthesis of the appealing compound 1,3-propanediol (1,3-PDO). This novel pathway is first confirmed in vitro using HCHO and pyruvate as substrates. It is then demonstrated in vivo in E. coli for 1,3-PDO production from HCHO and methanol with glucose as a cosubstrate. This de novo pathway has several decisive advantages over the known metabolic pathways for 1,3-PDO: (1) C1 carbon is directly channeled into a precursor of 1,3-PDO; (2) the use of pyruvate as an acceptor of HCHO is glycerol-independent, circumventing thus the need of coenzyme B12 as cofactor for glycerol dehydration; (3) the pathway is much shorter and more simple than the recently proposed l-homoserine-dependent pathway, thus avoiding complicated regulations involving precursors for essential amino acids. In addition to proof-of-concept we further improved the host strain by deleting a gene (frmA) responsible for the conversion of HCHO to formate, thereby increasing the production of 1,3-PDO from 298.3 ± 11.4 mg/L to 508.3 ± 9.1 mg/L and from 3.8 mg/L to 32.7 ± 0.8 mg/L with HCHO and methanol as cosubstrate of glucose fermentation, respectively. This work is the first study demonstrating a genetically engineered E. coli that can directly use HCHO or methanol for the synthesis of 2-keto-4-hydroxybutyrate and its further conversion to 1,3-PDO.

The Draft Genome Sequence of Methylophilus sp. D22, Capable of Growing Under High Concentration of Methanol.

In this study, a wild type Methylophilus sp. strain D22 belonging to the family Methylophilus was isolated and characterized, which shows high tolerance towards methanol, as it can grow under 50g/L of methanol. Methylophilus sp. strain D22 was isolated from the lake sludge in Nanjing Tech University, China. The assembled draft genome contains one circular chromosome with 3,004,398bp, 49.7% of GC content, and 2107 predicted encoding proteins. Sequence-based genomic analysis demonstrates that the assimilation pathway of ribulose monophosphate (RuMP) pathway and dissimilation pathway of tetrahydromethanopterin (H4MPT) pathway are co-existing and contribute to the high methanol utilization efficiency.

Species interactions and distinct microbial communities in high Arctic permafrost affected cryosols are associated with the CH4 and CO2 gas fluxes.

Microbial metabolism of the thawing organic carbon stores in permafrost results in a positive feedback loop of greenhouse gas emissions. CO2 and CH4 fluxes and the associated microbial communities in Arctic cryosols are important in predicting future warming potential of the Arctic. We demonstrate that topography had an impact on CH4 and CO2 flux at a high Arctic ice-wedge polygon terrain site, with higher CO2 emissions and lower CH4 uptake at troughs compared to polygon interior soils. The pmoA sequencing suggested that USCα cluster of uncultured methanotrophs is likely responsible for observed methane sink. Community profiling revealed distinct assemblages across the terrain at different depths. Deeper soils contained higher abundances of Verrucomicrobia and Gemmatimonadetes, whereas the polygon interior had higher Acidobacteria and lower Betaproteobacteria and Deltaproteobacteria abundances. Genome sequencing of isolates from the terrain revealed presence of carbon cycling genes including ones involved in serine and ribulose monophosphate pathways. A novel hybrid network analysis identified key members that had positive and negative impacts on other species. Operational Taxonomic Units (OTUs) with numerous positive interactions corresponded to Proteobacteria, Candidatus Rokubacteria and Actinobacteria phyla, while Verrucomicrobia and Acidobacteria members had negative impacts on other species. Results indicate that topography and microbial interactions impact community composition.

Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli

BackgroundMethanol has attracted increased attention as a non-food alternative carbon source to sugar for biological production of chemicals and fuels. Moreover, the high degree of reduction of methanol offers some advantages in increasing the production yields of NAD(P)H-dependent metabolites. Here, we demonstrate an example of methanol bioconversion with the aim of improving production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli.ResultsA synthetic methylotrophic E. coli was engineered with a nicotinamide adenine dinucleotide (NAD+)-dependent methanol dehydrogenase (MDH) and ribulose monophosphate (RuMP) pathway. Regarding the limited MDH activity, the role of activator proteins in vivo was investigated, and the NudF protein was identified capable of improving MDH activity and triggering increased methanol metabolism. Using 13C-methanol-labeling experiments, we confirmed methanol assimilation in the methylotrophic E. coli. A cycling RuMP pathway for methanol assimilation was also demonstrated by detecting multiple labeled carbons for several compounds. Finally, using the NAD(P)H-dependent metabolite lysine as a test, the potential of methanol bioconversion to generate value-added metabolites was determined. To further characterize the benefit of methanol as the carbon source, extra NADH from methanol oxidation was engineered to generate NADPH to improve lysine biosynthesis by expression of the POS5 gene from Saccharomyces cerevisiae, which resulted in a twofold improvement of lysine production. Moreover, this new sink further pulled upstream methanol utilization.ConclusionThrough engineering methanol metabolism, lysine biosynthesis, and NADPH regeneration pathway from NADH, the bioconversion of methanol to improve chemical synthesis was successfully achieved in methylotrophic E. coli.

Characterization of Rhodococcus wratislaviensis, a New Gram-Positive Facultative Methylotroph, and Properties of Its C1 Metabolism

A facultative methylotroph, strain 2AzMo (VKM Ac-2782), was isolated from the coastal zone of the Sea of Azov. The cells of the isolate are aerobic gram-positive nonmotile rods. Optimal growth occurs at 1% NaCl, 28°C, and pH 7.5 with 1% СH3OH or 0.3% methylamine as the carbon and energy sources. A broad spectrum of polycarbon substrates is also utilized. Sequencing of the 16S rRNA gene of the strain revealed its similarity to Rhodococcus species: 99.9% to R. wratislaviensis IEGM 1112T (=NCIMB 13082T), 99.4% to R. imtechensis IEGM 940T (=RKJ300T), and 99.2% to R. koreensis IEGM 962T (=DNP505T). The level of DNA–DNA homology of strain 2AzMo with R. wratislaviensis IEGM 1112T (=NCIMB 13082T) was 76%, supporting its identification as a strain of this species. However, unlike strain 2AzMo, the type strain R. wratislaviensis IEGM 1112T, as well as other members of this genus (R. imtechensis IEGM 940T, R. koreensis IEGM 962T, and R. opacus IEGM 716T), do not grow on methanol and methylamine. Methanol oxidation by R.wratislaviensis 2AzMo is catalyzed by alcohol dehydrogenase, which uses 4-nitroso-N,N-dimethylaniline as an artificial electron acceptor. Methylamine is oxidized by methylamine dehydrogenase and the enzymes of the N-methylglutamate pathway. Formaldehyde is then assimilated via the fructose bisphosphate aldolase variant of the ribulose monophosphate pathway of C1 metabolism. Ammonium is assimilated by α-ketoglutarate reductive amination and via the glutamate cycle.

Rare Earth Elements Alter Redox Balance in Methylomicrobium alcaliphilum 20ZR.

Background: Rare Earth Elements (REEs) control methanol utilization in both methane- and methanol-utilizing microbes. It has been established that the addition of REEs leads to the transcriptional repression of MxaFI-MeDH [a two-subunit methanol dehydrogenase (MeDH), calcium-dependent] and the activation of XoxF-MeDH (a one-subunit MeDH, lanthanum-dependent). Both enzymes are pyrroquinoline quinone-dependent alcohol dehydrogenases and show significant homology; however, they display different kinetic properties and substrate specificities. This study investigates the impact of the MxaFI to XoxF switch on the behavior of metabolic networks at a global scale.Results: In this study we investigated the steady-state growth of Methylomicrobium alcaliphilum 20ZR in media containing calcium (Ca) or lanthanum (La, a REE element). We found that cells supplemented with La show a higher growth rate compared to Ca-cultures; however, the efficiency of carbon conversion, estimated as biomass yield, is higher in cells grown with Ca. Three complementary global-omics approaches–RNA-seq transcriptomics, proteomics, and metabolomics–were applied to investigate the mechanisms of improved growth vs. carbon conversion. Cells grown with La showed the transcriptional activation of the xoxF gene, a homolog of the formaldehyde-activating enzyme (fae2), a putative transporter, genes for hemin-transport proteins, and nitrate reductase. In contrast, genes for mxaFI and associated cytochrome (mxaG) expression were downregulated. Proteomic profiling suggested additional adjustments of the metabolic network at the protein level, including carbon assimilation pathways, electron transport systems, and the tricarboxylic acid (TCA) cycle. Discord between gene expression and protein abundance changes points toward the possibility of post-transcriptional control of the related systems including key enzymes of the TCA cycle and a set of electron-transport carriers. Metabolomic data followed proteomics and showed the reduction of the ribulose-monophosphate (RuMP) pathway intermediates and the increase of the TCA cycle metabolites.Conclusion: Cells exposed to REEs display higher rates of growth but have lower carbon conversion efficiency compared to cells supplemented with Ca. The most plausible explanation for these physiological changes is an increased conversion of methanol into formate by XoxF-MeDH, which further stimulates methane oxidation but limits both the supply of reducing power and flux of formaldehyde into the RuMP pathway.

Metabolic Engineering of Escherichia coli for High Yield Production of Succinic Acid Driven by Methanol.

Methanol is increasingly becoming an attractive carbon feedstock for the production of various biochemicals due to its high abundance and low price. In this study, when methanol assimilation module was introduced into succinic acid producing Escherichia coli by employing the NAD-dependent methanol dehydrogenase from Bacillus methanolicus and ribulose monophosphate pathway from different donor organisms, succinic acid yield was increased from 0.91 ± 0.08 g/g to 0.98 ± 0.11 g/g with methanol as an auxiliary substrate under the anaerobic fermentation. Further 13C-labeling experiments showed that the recombinant strain successfully converted methanol into succinic acid, as the carbon atom of carboxyl group in succinic acid was labeled by 13C. It was found that the NADH generated by methanol oxidation would benefit succinate production, as the NADH/NAD+ ratio in vivo was decreased from 0.67 to 0.45 in the engineered strain, indicating that the efficiency of succinic acid synthesis was significantly improved when driven by methanol. This study represents a successful case for the development of reducing chemical production using methanol as an auxiliary substrate.