Methylotrophic Microorganisms Research Articles

Identification and characterization of a novel formaldehyde dehydrogenase in Bacillus subtilis.

Formaldehyde is a key metabolite in methanol assimilation for many methylotrophic microorganisms, and at the same time, it is toxic to all living cells, which means its intracellular concentrations must be tightly controlled. An in-depth understanding of methanol detoxification systems in industrially relevant microorganisms is a prerequisite for the introduction of methanol utilization pathways into their metabolism (synthetic methylotrophy). Bacillus subtilis, an industrial workhorse conventionally used for the production of enzymes, is known to possess two formaldehyde detoxification pathways. Here, we identify a novel formaldehyde dehydrogenase in this bacterium as a path towards creating innovative prospect strategies for strain engineering towards synthetic methylotrophy.

Formaldehyde: An Essential Intermediate for C1 Metabolism and Bioconversion.

Formaldehyde is an intermediate metabolite of methylotrophic microorganisms that can be obtained from formate and methanol through oxidation-reduction reactions. Formaldehyde is also a one-carbon (C1) compound with high uniquely reactive activity and versatility, which is more amenable to further biocatalysis. Biosynthesis of high-value-added chemicals using formaldehyde as an intermediate is theoretically feasible and promising. This review focuses on the design of the biosynthesis of high-value-added chemicals using formaldehyde as an essential intermediate. The upstream biosynthesis and downstream bioconversion pathways of formaldehyde as an intermediate metabolite are described in detail, aiming to highlight the important role of formaldehyde in the transition from inorganic to organic carbon and carbon chain elongation. In addition, challenges and future directions of formaldehyde as an intermediate for the chemicals are discussed, with the expectation of providing ideas for the utilization of C1.

Microbial communities mediating net methylmercury formation along a trophic gradient in a peatland chronosequence.

Peatlands are generally important sources of methylmercury (MeHg) to adjacent aquatic ecosystems, increasing the risk of human and wildlife exposure to this highly toxic compound. While microorganisms play important roles in mercury (Hg) geochemical cycles where they directly and indirectly affect MeHg formation in peatlands, potential linkages between net MeHg formation and microbial communities involving these microorganisms remain unclear. To address this gap, microbial community composition and specific marker gene transcripts were investigated along a trophic gradient in a geographically constrained peatland chronosequence. Our results showed a clear spatial pattern in microbial community composition along the gradient that was highly driven by peat soil properties and significantly associated with net MeHg formation as approximated by MeHg concentration and %MeHg of total Hg concentration. Known fermentative, syntrophic, methanogenic and iron-reducing metabolic guilds had the strong positive correlations to net MeHg formation, while methanotrophic and methylotrophic microorganisms were negatively correlated. Our results indicated that sulfate reducers did not have a key role in net MeHg formation. Microbial activity as interpreted from 16S rRNA sequences was significantly correlated with MeHg and %MeHg. Our findings shed new light on the role of microbial community in net MeHg formation of peatlands that undergo ontogenetic change.

Methylotrophic bacteria with cobalamin-dependent mutases in primary metabolism as potential strains for vitamin B12 production

Several bacterial species are known for their ability to synthesize vitamin B12 but biotechnological vitamin B12 production today is restricted to Pseudomonas denitrificans and Propionibacterium freudenreichii. Nevertheless, the rising popularity of veganism leads to a growing demand for vitamin B12 and thereby interest in alternative strains which can be used as efficient vitamin B12 sources. In this work, we demonstrate that methylotrophic microorganisms which utilize the ethylmalonyl-CoA pathway containing B12-dependent enzymes are capable of active vitamin B12 production. Several bacteria with an essential function of the pathway were tested for vitamin B12 synthesis. Among the identified strains, Hyphomicrobium sp. DSM3646 demonstrated the highest vitamin B12 levels reaching up to 17.9 ± 5.05 µg per g dry cell weight. These relatively high vitamin B12 concentrations achieved in simple cultivation experiments were performed in a mineral methanol medium, which makes Hyphomicrobium sp. DSM3646 a new promising cobalamin-producing strain.

Overview of methylotrophic microorganisms in agriculture

Overview of methylotrophic microorganisms in agriculture

Methylotrophic bacterium-based molecular sensor for the detection of low concentrations of methanol

Methylotrophic bacterium Methylorubrum extorquens is a promising microorganism for the production of value-added compounds from methanol. This study focused on the development of a single-cell level biosensor system that detects methanol by using the intrinsic regulatory machinery which responds to the presence of methanol in this bacterium. A green fluorescent protein (GFP) gene located downstream of the promoter region of the serine glyoxylate aminotransferase gene (Psga) or the methanol dehydrogenase subunit 1 precursor gene (PmxaF) was inserted into the chromosome of M.extorquens wild-type strain AM1. The expression of GFP upon methanol exposure was measured by spectrofluorometer and fluorescence-activated cell sorting (FACS). The strain harboring Psga-gfp emitted fluorescence only when methanol was supplied to the culture medium, while the other strain harboring PmxaF-gfp showed high basal fluorescence even in the absence of methanol. The fluorescence intensity of the Psga-gfp strain depended on a methanol concentration higher than 25μM, and the sensitivity and dose-dependency of this strain were much higher than previous systems using Escherichia coli. The methanol-sensing properties of the engineered M.extorquens strain were comparable to those of a methylotrophic yeast-based biosensor, suggesting the usefulness of methylotrophic microorganisms as platforms for single-cell sensing of C1 compounds. The constructed methanol sensor strain, coupled with flow cytometry techniques, provides a high-throughput and highly sensitive screening method for the selection of functional methanol-producing enzymes.

Release of formaldehyde during the biofiltration of methanol vapors in a peat biofilter inoculated with Pichia pastoris GS115

Methanol can be effectively removed from air by biofiltration (Shareefdeen et al., 1993; Babbitt et al., 2009 [1,2]). However, formaldehyde is one of the first metabolic intermediates in the consumption of methanol in methylotrophic microorganisms (Negruţa et al., 2010 [3]), and it can be released out of the cell constituting a secondary emission. The total removal of methanol was achieved up to input loads of 263 g m−3 h−1 and the maximum elimination capacity of the system was obtained at an empty bed residence times of 90 s and reached 330 g m−3 h−1 at an input methanol load of 414 g m−3 h−1 and 80% of removal efficiency. Formaldehyde was detected inside the biofilter when the input methanol load was above 212 g m−3 h−1. Biomass in the filter bed was able to degrade the formaldehyde generated, but with the increase of the methanol input load, the unconsumed formaldehyde was released outside the biofilter. The maximum concentration registered at the output of the system was 3.98 g m−3 when the methanol load was 672 g m−3 h−1 in an empty bed residence times of 60 s. Formaldehyde is produced inside a biofilter when methanol is treated in a biofiltration system inoculated with Pichia pastoris. Biomass present in the reactor is capable of degrading the formaldehyde generated as the concentration of methanol decreases. However, high methanol loads can lead to the generation and release of formaldehyde into the environment. How to cite: Guerrero K, Arancibia A, Caceres M, et al. Release of formaldehyde during the biofiltration of methanol vapors in a peat biofilter inoculated with Pichia pastoris GS115. Electron J Biotechnol 2019;40. https://doi.org/10.1016/j.ejbt.2019.04.003.

Communal metabolism by Methylococcaceae and Methylophilaceae is driving rapid aerobic methane oxidation in sediments of a shallow seep near Elba, Italy.

The release of abiotic methane from marine seeps into the atmosphere is a major source of this potent greenhouse gas. Methanotrophic microorganisms in methane seeps use methane as carbon and energy source, thus significantly mitigating global methane emissions. Here, we investigated microbial methane oxidation at the sediment-water interface of a shallow marine methane seep. Metagenomics and metaproteomics, combined with 13 C-methane stable isotope probing, demonstrated that various members of the gammaproteobacterial family Methylococcaceae were the key players for methane oxidation, catalysing the first reaction step to methanol. We observed a transfer of carbon to methanol-oxidizing methylotrophs of the betaproteobacterial family Methylophilaceae, suggesting an interaction between methanotrophic and methylotrophic microorganisms that allowed for rapid methane oxidation. From our microcosms, we estimated methane oxidation rates of up to 871 nmol of methane per gram sediment per day. This implies that more than 50% of methane at the seep is removed by microbial oxidation at the sediment-water interface, based on previously reported in situ methane fluxes. The organic carbon produced was further assimilated by different heterotrophic microbes, demonstrating that the methane-oxidizing community supported a complex trophic network. Our results provide valuable eco-physiological insights into this specialized microbial community performing an ecosystem function of global relevance.

Release of formaldehyde during the biofiltration of methanol vapors in a peat biofilter inoculated with Pichia pastoris GS115

Methanol can be effectively removed from air by biofiltration. However, formaldehyde is one of the first metabolic intermediates in the consumption of methanol in methylotrophic microorganisms, and it can be released out of the cell constituting a secondary emission.

Methylotrophic Yeasts: Current Understanding of Their C1-Metabolism and its Regulation by Sensing Methanol for Survival on Plant Leaves.

Methylotrophic yeasts, which are able to utilize methanol as the sole carbon and energy source, have been intensively studied in terms of physiological function and practical applications. When these yeasts grow on methanol, the genes encoding enzymes and proteins involved in methanol metabolism are strongly induced. Simultaneously, peroxisomes, organelles that contain the key enzymes for methanol metabolism, massively proliferate. These characteristics have made methylotrophic yeasts efficient hosts for heterologous protein production using strong and methanol-inducible gene promoters and also model organisms for the study of peroxisome dynamics. Much attention has been paid to the interaction between methylotrophic microorganisms and plants. In this chapter, we describe how methylotrophic yeasts proliferate and survive on plant leaves, focusing on their physiological functions and lifestyle in the phyllosphere. Our current understanding of the molecular basis of methanol-inducible gene expression, including methanol-sensing and its applications, is also summarized.

Characterization of a novel thermotolerant NAD+-dependent formate dehydrogenase from hot climate plant cotton (Gossypium hirsutum L.).

NAD+-dependent formate dehydrogenases (FDH, EC 1.2.1.2), providing energy to the cell in methylotrophic microorganisms, are stress proteins in higher plants and the level of FDH expression increases under several abiotic and biotic stress conditions. They are biotechnologically important enzymes in NAD(P)H regeneration as well as CO2 reduction. Here, the truncated form of the Gossypium hirsutum fdh1 cDNA was cloned into pQE-2 vector, and overexpressed in Escherichia coli DH5α-T1 cells. Recombinant GhFDH1 was purified 26.3-fold with a yield of 87.3%. Optimum activity was observed at pH 7.0, when substrate is formate. Kinetic analyses suggest that GhFDH1 has considerably high affinity to formate (0.76 ± 0.07mM) and NAD+ (0.06 ± 0.01mM). At the same time, the affinity (1.98 ± 0.4mM) and catalytic efficiency (0.0041) values of the enzyme for NADP+ show that GhFDH1 is a valuable enzyme for protein engineering studies that is trying to change the coenzyme preference from NAD to NADP which has a much higher cost than that of NAD. Improving the NADP specificity is important for NADPH regeneration which is an important coenzyme used in many biotechnological production processes. The Tm value of GhFDH1 is 53.3°C and the highest enzyme activity is measured at 30°C with a half-life of 61h. Whilst further improvements are still required, the obtained results show that GhFDH1 is a promising enzyme for NAD(P)H regeneration for its prominent thermostability and NADP+ specificity.

Expression of heterologous non-oxidative pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli methylotroph

Synthetic methylotrophy aims to develop non-native methylotrophic microorganisms to utilize methane or methanol to produce chemicals and biofuels. We report two complimentary strategies to further engineer a previously engineered methylotrophic E. coli strain for improved methanol utilization. First, we demonstrate improved methanol assimilation in the presence of small amounts of yeast extract by expressing the non-oxidative pentose phosphate pathway (PPP) from Bacillus methanolicus. Second, we demonstrate improved co-utilization of methanol and glucose by deleting the phosphoglucose isomerase gene (pgi), which rerouted glucose carbon flux through the oxidative PPP. Both strategies led to significant improvements in methanol assimilation as determined by 13C-labeling in intracellular metabolites. Introduction of an acetone-formation pathway in the pgi-deficient methylotrophic E. coli strain led to improved methanol utilization and acetone titers during glucose fed-batch fermentation.

Production of 2-Hydroxyisobutyric Acid from Methanol by Methylobacterium extorquens AM1 Expressing (R)-3-Hydroxybutyryl Coenzyme A-Isomerizing Enzymes.

In this study, we genetically modified a methylotrophic bacterium in order to channel intermediates of its overflow metabolism to the C4 carboxylic acid 2-hydroxyisobutyric acid, a precursor of acrylic glass. This has implications for biotechnology, as it shows that reduced C1 substrates, such as methanol and formic acid, can be alternative feedstocks for producing today's commodities. We found that product titers and yields depend more on host physiology than on the activity of the introduced heterologous function modifying the overflow metabolism. In addition, we show that the fitness of recombinant strains substantially varies when they express orthologous genes from different origins. Further studies are needed to extend the overflow production phase in methylotrophic microorganisms for the implementation of biotechnological processes.

SOIL ANALYSIS ON MICROBIOLOGICAL INDICATOR VALUES IN THE AREA OF UNDERGROUND NATURE GAS STORAGE

The aim is to conduct the biodiagnosis of soil sampled in the area of the underground storage of natural gas (UGS) (Stepnoye village, Saratov region) with the use of microbiological analysis. Methods. In the course of the work, using Koch’s solid medium method were estimated the following: the total number of heterotrophic microorganisms on meat peptone agar, amount of methylotrophic, hydrocarbon oxidizing, ironoxidizing and sulfur-oxidizing bacteria in the respective selective mediums; as well as the hydrogen index, oxidation-reduction potential and magnetic susceptibility of the soil. Results. Microbiological analysis of the soil in the Stepnovsky UGS area has shown changes in soil biocenoses. A reduced content of heterotrophic microorganisms was detected in comparison with background samples. In a number of samples, we also discovered an increased content of hydrocarbon-oxidizing and methylotrophic microorganisms, including obligate ones, which suggest the methane entering the upper layers of the soil. An increased content of sulfur-oxidizing bacteria was also detected; the presence of iron-oxidizing microorganisms in the soil was shown. Conclusions. The revealed features in the content of indicator microorganisms in the soil sampled in the area of the underground storage of natural gas, indicating the development of specialized soil microorganisms, confirmed the presence of contaminants in the soil which are hydrocarbons and sulfur compounds. The use of microbiological indicator values seems promising for environmental monitoring of soils in the areas of gas storage and early detection of methane leakage.

Culturable Facultative Methylotrophic Bacteria from the Cactus Neobuxbaumia macrocephala Possess the Locus xoxF and Consume Methanol in the Presence of Ce3+ and Ca2.

Methanol-consuming culturable bacteria were isolated from the plant surface, rhizosphere, and inside the stem of Neobuxbaumia macrocephala. All 38 isolates were facultative methylotrophic microorganisms. Their classification included the Classes Actinobacteria, Sphingobacteriia, Alpha-, Beta-, and Gammaproteobacteria. The deduced amino acid sequences of methanol dehydrogenase obtained by PCR belonging to Actinobacteria, Alpha-, Beta-, and Gammaproteobacteria showed high similarity to rare-earth element (REE)-dependent XoxF methanol dehydrogenases, particularly the group XoxF5. The sequences included Asp301, the REE-coordinating amino acid, present in all known XoxF dehydrogenases and absent in MxaF methanol dehydrogenases. The quantity of the isolates showed positive hybridization with a xoxF probe, but not with a mxaF probe. Isolates of all taxonomic groups showed methylotrophic growth in the presence of Ce3+ or Ca2+. The presence of xoxF-like sequences in methylotrophic bacteria from N. macrocephala and its potential relationship with their adaptability to xerophytic plants are discussed.

Device and method for eliminating NOx and N2O

Some strains belonging to Pseudomonas putida have been found to degrade formaldehyde, making them promising methylotrophic bacteria in the treatment of environmental formaldehyde. However, the pathway involved in formaldehyde degradation of P. putida has not been extensively investigated. P. putida IOFA1, a strain isolated from the deep-sea sediments of the Indian Ocean, was found to be extremely tolerant of formaldehyde (up to 10 mM), efficiently degrading formaldehyde. In this study, the transcriptome profiles of the IOFA1 strain at different stages of formaldehyde degradation were characterized. A total of 709 genes were found to be significantly altered during formaldehyde degradation. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis showed that methanol metabolism, protein synthesis, and material transportation were highly enriched during formaldehyde degradation. The expression pattern of key methanol metabolism genes, which may be responsible for formaldehyde degradation, was investigated further with the quantitative real-time chain reaction (qRT-PCR) method. Our results showed that the IOFA1 strain may adopt a metabolic strategy of formaldehyde degradation different from other reported methylotrophic microorganisms. Therefore, our study presents a novel insight into the mechanism of formaldehyde degradation, which may help in engineering P. putida strains with a greater ability of degrading formaldehyde.

Pseudomonas putida IOFA1 transcriptome profiling reveals a metabolic pathway involved in formaldehyde degradation

Some strains belonging to Pseudomonas putida have been found to degrade formaldehyde, making them promising methylotrophic bacteria in the treatment of environmental formaldehyde. However, the pathway involved in formaldehyde degradation of P. putida has not been extensively investigated. P. putida IOFA1, a strain isolated from the deep-sea sediments of the Indian Ocean, was found to be extremely tolerant of formaldehyde (up to 10mM), efficiently degrading formaldehyde. In this study, the transcriptome profiles of the IOFA1 strain at different stages of formaldehyde degradation were characterized. A total of 709 genes were found to be significantly altered during formaldehyde degradation. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis showed that methanol metabolism, protein synthesis, and material transportation were highly enriched during formaldehyde degradation. The expression pattern of key methanol metabolism genes, which may be responsible for formaldehyde degradation, was investigated further with the quantitative real-time chain reaction (qRT-PCR) method. Our results showed that the IOFA1 strain may adopt a metabolic strategy of formaldehyde degradation different from other reported methylotrophic microorganisms. Therefore, our study presents a novel insight into the mechanism of formaldehyde degradation, which may help in engineering P. putida strains with a greater ability of degrading formaldehyde.

Culture-dependent and culture-independent methods reveal diverse methylotrophic communities in terrestrial environments.

One-carbon compounds such as methanol, dimethylsulfide (DMS) and dimethylsulfoxide (DMSO) are significant intermediates in biogeochemical cycles. They are suggested to affect atmospheric chemistry and global climate. Methylotrophic microorganisms are considered as a significant sink for these compounds; therefore, we analyzed the diversity of terrestrial bacteria that utilize methanol, DMS and DMSO as carbon and energy source using culture-dependent and culture-independent methods. The effect of habitat type on the methylotrophic community structure was also investigated in rhizosphere and bulk soil. While thirteen strains affiliated to the genera Hyphomicrobium, Methylobacterium, Pseudomonas, Hydrogenophaga, Rhodococcus, Flavobacterium and Variovorax were isolated, denaturing gradient gel electrophoresis revealed the dominance of Thiobacillus, Rhodococcus, Flavobacterium and Bacteroidetes species. Furthermore, methylotrophic communities that degrade methanol or DMS are not shaped by terrestrial habitat type. Rhizosphere and soil samples showed dominance of Methylophilus spp. and Methylovorus spp. for methanol enrichments; Cytophaga spp., Pseudomonas tremae and Thiobacillus thioparus for DMS enrichments.

Methylobacterium extorquens: methylotrophy and biotechnological applications.

Methylotrophy is the ability to use reduced one-carbon compounds, such as methanol, as a single source of carbon and energy. Methanol is, due to its availability and potential for production from renewable resources, a valuable feedstock for biotechnology. Nature offers a variety of methylotrophic microorganisms that differ in their metabolism and represent resources for engineering of value-added products from methanol. The most extensively studied methylotroph is the Alphaproteobacterium Methylobacterium extorquens. Over the past five decades, the metabolism of M. extorquens has been investigated physiologically, biochemically, and more recently, using complementary omics technologies such as transcriptomics, proteomics, metabolomics, and fluxomics. These approaches, together with a genome-scale metabolic model, facilitate system-wide studies and the development of rational strategies for the successful generation of desired products from methanol. This review summarizes the knowledge of methylotrophy in M. extorquens, as well as the available tools and biotechnological applications.

Sulfur oxidizers dominate carbon fixation at a biogeochemical hot spot in the dark ocean

Bacteria and archaea in the dark ocean (>200 m) comprise 0.3-1.3 billion tons of actively cycled marine carbon. Many of these microorganisms have the genetic potential to fix inorganic carbon (autotrophs) or assimilate single-carbon compounds (methylotrophs). We identified the functions of autotrophic and methylotrophic microorganisms in a vent plume at Axial Seamount, where hydrothermal activity provides a biogeochemical hot spot for carbon fixation in the dark ocean. Free-living members of the SUP05/Arctic96BD-19 clade of marine gamma-proteobacterial sulfur oxidizers (GSOs) are distributed throughout the northeastern Pacific Ocean and dominated hydrothermal plume waters at Axial Seamount. Marine GSOs expressed proteins for sulfur oxidation (adenosine phosphosulfate reductase, sox (sulfur oxidizing system), dissimilatory sulfite reductase and ATP sulfurylase), carbon fixation (ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO)), aerobic respiration (cytochrome c oxidase) and nitrogen regulation (PII). Methylotrophs and iron oxidizers were also active in plume waters and expressed key proteins for methane oxidation and inorganic carbon fixation (particulate methane monooxygenase/methanol dehydrogenase and RuBisCO, respectively). Proteomic data suggest that free-living sulfur oxidizers and methylotrophs are among the dominant primary producers in vent plume waters in the northeastern Pacific Ocean.