Microbial Stabilization and Kinetic Enhancement of Marine Methane Hydrates

Abstract

In clathrate hydrates, a water host lattice encages small guest molecules in cavities. Methane hydrates are the most widespread in-situ clathrate in the permafrost and continental-shelf ocean regions, constituting a significant energy resource, and prompting recent marine-hydrate gas-production trials. Despite exciting engineering advances and a few marine-mimicking laboratory studies of methane-hydrate kinetics and stabilization, from microbial perspectives, little is known about a potential microbial origin of marine hydrates, nor their possible formation kinetics or potential stabilization by microbial sources. Here, for the first time, we show that an exported, extra-cytoplasmic porin – produced by a marine methylotrophic bacterium culture – provides the basis for kinetic enhancement and stabilization of methane hydrates under conditions simulating the seabed environment. We then identify the key protein at play, and we therefore suggest microbe-based stabilization of marine hydrates is evidently a property likely to be found in many marine bacteria. Our research opens the possibility of managing marine-hydrate deposits using microbiological strategies for environmental and societal benefit.     

Engineering Escherichia coli for methanol-dependent growth on glucose for metabolite production

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methanol auxotrophs have proved successful, these studies focused on scarce and expensive co-substrates. Here, we engineered E. coli for methanol-dependent growth on glucose, an abundant and inexpensive co-substrate, via deletion of glucose 6-phosphate isomerase (pgi), phosphogluconate dehydratase (edd), and ribose 5-phosphate isomerases (rpiAB). Since the parental strain did not exhibit methanol-dependent growth on glucose in minimal medium, we first achieved methanol-dependent growth via amino acid supplementation and used this medium to evolve the strain for methanol-dependent growth in glucose minimal medium. The evolved strain exhibited a maximum growth rate of 0.15 h⁻¹ in glucose minimal medium with methanol, which is comparable to that of other synthetic methanol auxotrophs. Whole genome sequencing and ¹³C-metabolic flux analysis revealed the causative mutations in the evolved strain. A mutation in the phosphotransferase system enzyme I gene (ptsI) resulted in a reduced glucose uptake rate to maintain a one-to-one molar ratio of substrate utilization. Deletion of the e14 prophage DNA region resulted in two non-synonymous mutations in the isocitrate dehydrogenase (icd) gene, which reduced TCA cycle carbon flux to maintain the internal redox state. In high cell density glucose fed-batch fermentation, methanol-dependent acetone production resulted in 22% average carbon labeling of acetone from ¹³C-methanol, which far surpasses that of the previous best (2.4%) found with methylotrophic E. coli Δpgi. This study addresses the need to identify appropriate co-substrates for engineering synthetic methanol auxotrophs and provides a basis for the next steps toward industrial one-carbon bioprocessing.     

Mixing and matching methylotrophic enzymes to design a novel methanol utilization pathway in E. coli

One-carbon (C1) compounds, such as methanol, have recently gained attention as alternative low-cost and non-food feedstocks for microbial bioprocesses. Considerable research efforts are thus currently focused on the generation of synthetic methylotrophs by transferring methanol assimilation pathways into established bacterial production hosts. In this study, we used an iterative combination of dry and wet approaches to design, implement and optimize this metabolic trait in the most common chassis, E. coli. Through in silico modelling, we designed a new route that “mixed and matched” two methylotrophic enzymes: a bacterial methanol dehydrogenase (Mdh) and a dihydroxyacetone synthase (Das) from yeast. To identify the best combination of enzymes to introduce into E. coli, we built a library of 266 pathway variants containing different combinations of Mdh and Das homologues and screened it using high-throughput ¹³C-labeling experiments. The highest level of incorporation of methanol into central metabolism intermediates (e.g. 22% into the PEP), was obtained using a variant composed of a Mdh from A. gerneri and a codon-optimized version of P. angusta Das. Finally, the activity of this new synthetic pathway was further improved by engineering strategic metabolic targets identified using omics and modelling approaches. The final synthetic strain had 1.5 to 5.9 times higher methanol assimilation in intracellular metabolites and proteinogenic amino acids than the starting strain did. Broadening the repertoire of methanol assimilation pathways is one step further toward synthetic methylotrophy in E. coli.     

Methane-based symbiosis in a mussel, Bathymodiolus platifrons, from cold seeps in Sagami Bay, Japan

Abstract. Bathymodiolus platifrons , a chemosynthetic mussel from cold seeps off Japan, relies for its nutrition on the productivity of methylotrophic or methanotrophic endosymbionts. High densities of bacterial symbionts appearing to be type I methanotrophs were observed in transmission electron micrographs of gill tissues. Methanol dehydrogenase activity in gill tissue from a single individual was positive compared to non-methanotrophic control samples, indicating a high potential for methanotrophy. Stable isotopic ratios of carbon in symbiont-containing gill tissue, as well as host tissues, were extremely depleted in ¹³C, and similar to values reported for other methanotrophic species. TEMs of gill tissue showing symbionts in various stages of digestion support the hypothesis that carbon transfer from symbionts to B. platifrons occurs through intracellular digestion of the symbionts. Discovery of methane- or methanolbased symbioses in B. platifrons from cold seeps in Sagami Bay extends the range of such symbioses to include cold seeps and hydrothermal vents, and supports the idea that environmental methane levels control the distribution of these symbioses.     

Triggering the stringent response enhances synthetic methanol utilization in Escherichia coli

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methylotrophs have proved successful, autonomous methylotrophy, i.e. the ability to utilize methane or methanol as sole carbon and energy substrates, has not yet been realized. Here, we address an important limitation of autonomous methylotrophy in E. coli: the inability of the organism to synthesize several amino acids when grown on methanol. By activating the stringent/stress response via ppGpp overproduction, or DksA and RpoS overexpression, we demonstrate improved biosynthesis of proteinogenic amino acids via endogenous upregulation of amino acid synthesis pathway genes. Thus, we were able to achieve biosynthesis of several limiting amino acids from methanol-derived carbon, in contrast to the control methylotrophic E. coli strain. This study addresses a key limitation currently preventing autonomous methylotrophy in E. coli and possibly other synthetic methylotrophs and provides insight as to how this limitation can be alleviated via stringent/stress response activation.     

甲基营养菌MP688基因组完成图构建

目的:在Solexa测序建立的甲基营养菌MP688基因组精细图的基础上,构建MP688基因组完成图。方法:根据MP688基因组序列设计引物,进行常规PCR扩增,填补Scaffold内部缺口;利用BLASTN、BLAT软件分析预测6个Scaffold定位关系;通过组合PCR、长距离PCR等填充Scaffold间的物理缺口。结果:常规PCR填充Scaffold内部4个缺口;BLASTN、BLAT软件分析预测了Scaffold定位关系;组合PCR填充Scaffold间的2个物理缺口,长距离PCR填充其余4个物理缺口。结论:得到了MP688的基因组完成图,为MP688的吡咯喹啉醌生物合成途径的阐明和进一步的代谢工程育种奠定了基础。     

ASYMMETRIC, BIMODAL TRADE-OFFS DURING ADAPTATION OF METHYLOBACTERIUM TO DISTINCT GROWTH SUBSTRATES

Trade-offs between selected and nonselected environments are often assumed to exist during adaptation. This phenomenon is prevalent in microbial metabolism, where many organisms have come to specialize on a narrow breadth of substrates. One well-studied example is methylotrophic bacteria that can use single-carbon (C₁) compounds as their sole source of carbon and energy, but generally use few, if any, multi-C compounds. Here, we use adaptation of experimental populations of the model methylotroph, Methylobacterium extorquens AM1, to C₁ (methanol) or multi-C (succinate) compounds to investigate specialization and trade-offs between these two metabolic lifestyles. We found a general trend toward trade-offs during adaptation to succinate, but this was neither universal nor showed a quantitative relationship with the extent of adaptation. After 1500 generations, succinate-evolved strains had a remarkably bimodal distribution of fitness values on methanol: either an improvement comparable to the strains adapted on methanol or the complete loss of the ability to grow on C₁ compounds. In contrast, adaptation to methanol resulted in no such trade-offs. Based on the substantial, asymmetric loss of C₁ growth during growth on succinate, we suggest that the long-term maintenance of C₁ metabolism across the genus Methylobacterium requires relatively frequent use of C₁ compounds to prevent rapid loss.     

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

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

An “energy-auxotroph” Escherichia coli provides an in vivo platform for assessing NADH regeneration systems

An efficient in vivo regeneration of the primary cellular resources NADH and ATP is vital for optimizing the production of value-added chemicals and enabling the activity of synthetic pathways. Currently, such regeneration routes are tested and characterized mainly in vitro before being introduced into the cell. However, in vitro measurements could be misleading as they do not reflect enzyme activity under physiological conditions. Here, we construct an in vivo platform to test and compare NADH regeneration systems. By deleting dihydrolipoyl dehydrogenase in Escherichia coli, we abolish the activity of pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. When cultivated on acetate, the resulting strain is auxotrophic to NADH and ATP: acetate can be assimilated via the glyoxylate shunt but cannot be oxidized to provide the cell with reducing power and energy. This strain can, therefore, serve to select for and test different NADH regeneration routes. We exemplify this by comparing several NAD-dependent formate dehydrogenases and methanol dehydrogenases. We identify the most efficient enzyme variants under in vivo conditions and pinpoint optimal feedstock concentrations that maximize NADH biosynthesis while avoiding cellular toxicity. Our strain thus provides a useful platform for comparing and optimizing enzymatic systems for cofactor regeneration under physiological conditions.     

Development of a formaldehyde biosensor with application to synthetic methylotrophy

Formaldehyde is a prevalent environmental toxin and a key intermediate in single carbon metabolism. The ability to monitor formaldehyde concentration is, therefore, of interest for both environmental monitoring and for metabolic engineering of native and synthetic methylotrophs, but current methods suffer from low sensitivity, complex workflows, or require expensive analytical equipment. Here we develop a formaldehyde biosensor based on the FrmR repressor protein and cognate promoter of Escherichia coli. Optimization of the native repressor binding site and regulatory architecture enabled detection at levels as low as 1 µM. We then used the sensor to benchmark the in vivo activity of several NAD‐dependent methanol dehydrogenase (Mdh) variants, the rate‐limiting enzyme that catalyzes the first step of methanol assimilation. In order to use this biosensor to distinguish individuals in a mixed population of Mdh variants, we developed a strategy to prevent cross‐talk by using glutathione as a formaldehyde sink to minimize intercellular formaldehyde diffusion. Finally, we applied this biosensor to balance expression of mdh and the formaldehyde assimilation enzymes hps and phi in an engineered E. coli strain to minimize formaldehyde build‐up while also reducing the burden of heterologous expression. This biosensor offers a quick and simple method for sensitively detecting formaldehyde, and has the potential to be used as the basis for directed evolution of Mdh and dynamic formaldehyde control strategies for establishing synthetic methylotrophy.     

Microbial Methylotrophic Metabolism: Recent Metabolic Modeling Efforts and Their Applications In Industrial Biotechnology

Developing methylotrophic bacteria into cell factories that meet the chemical demand of the future could be both economical and environmentally friendly. Methane is not only an abundant, low‐cost resource but also a potent greenhouse gas, the capture of which could help to reduce greenhouse gas emissions. Rational strain design workflows rely on the availability of carefully combined knowledge often in the form of genome‐scale metabolic models to construct high‐producer organisms. In this review, the authors present the most recent genome‐scale metabolic models in aerobic methylotrophy and their applications. Further, the authors present models for the study of anaerobic methanotrophy through reverse methanogenesis and suggest organisms that may be of interest for expanding one‐carbon industrial biotechnology. Metabolic models of methylotrophs are scarce, yet they are important first steps toward rational strain‐design in these organisms.     

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

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

Recent advances toward the bioconversion of methane and methanol in synthetic methylotrophs

Abundant natural gas reserves, along with increased biogas production, have prompted recent interest in harnessing methane as an industrial feedstock for the production of liquid fuels and chemicals. Methane can either be used directly for fermentation or first oxidized to methanol via biological or chemical means. Methanol is advantageous due to its liquid state under normal conditions. Methylotrophy, defined as the ability of microorganisms to utilize reduced one-carbon compounds like methane and methanol as sole carbon and energy sources for growth, is widespread in bacterial communities. However, native methylotrophs lack the extensive and well-characterized synthetic biology toolbox of platform microorganisms like Escherichia coli, which results in slow and inefficient design-build-test cycles. If a heterologous production pathway can be engineered, the slow growth and uptake rates of native methylotrophs generally limit their industrial potential. Therefore, much focus has been placed on engineering synthetic methylotrophs, or non-methylotrophic platform microorganisms, like E. coli, that have been engineered with synthetic methanol utilization pathways. These platform hosts allow for rapid design-build-test cycles and are well-suited for industrial application at the current time. In this review, recent progress made toward synthetic methylotrophy (including methanotrophy) is discussed. Specifically, the importance of amino acid metabolism and alternative one-carbon assimilation pathways are detailed. A recent study that has achieved methane bioconversion to liquid chemicals in a synthetic E. coli methanotroph is also briefly discussed. We also discuss strategies for the way forward in order to realize the industrial potential of synthetic methanotrophs and methylotrophs.     

Breakdown of dimethyl sulphide by mixed cultures and by Thiobacillus thioparus

Abstract Dimethyl sulphide (DMS) was degraded by acclimatized activated sludge and by a mixed culture of Thiobacillus thioparus TK-1 and Pseudomonas sp. AK-2. While both these organisms persisted in stable co-culture on DMS, it was found that T. thioparus TK-1 and the derived strain TK-m grew in pure culture on DMS, and oxidized DMS with an apparent K _m of 4.5 × 10⁻⁵ M. During growth, all the DMS-sulphur was oxidized stoichiometrically to sulphate but no methanol was detected in pure cultures of TK-m. DMS-carbon was probably converted to CO₂, since the fixation of ¹⁴CO₂ was progressively diluted during growth of a culture on ¹⁴CO₂ and DMS. Growth yields were consistent with autotrophic growth, dependent on the oxidation of the methyl residues to CO₂ (probably with formaldehyde as a first intermediate) and the sulphide to sulphate. The organism thus appears to exhibit a mixture, from the one substrate, of chemolithotrophic and methylotrophic energy generation supporting autotrophic growth with CO₂ fixation.     

Metabolic characteristics of an aerobe isolated from a methylotrophic methanogenic enrichment culture

An anaerobic methylotrophic methanogenic enrichment culture, with sustained metabolic characteristics, including that of methanation for over a decade, was the choice of the present study on interspecies interactions. Growth and methanation by the enrichment were suppressed in the presence of antibiotics, and no methanogen grown on methanol could be isolated using stringent techniques. The present study confirmed syntrophic metabolic interactions in this enrichment with the isolation of a strain ofPseudomonas sp. The organism had characteristic metabolic versatility in metabolizing a variety of substrates including alcohols, aliphatic acids, amino acids, and sugars. Anaerobic growth was favoured with nitrate in the growth medium. Cells grown anaerobically with methanol, revealed maximal nitrate reductase activity. Constitutive oxidative activity of the membrane system emerged from the high-specific oxygen uptake and nitrate reductase activities of the aerobically and anerobically grown cells respectively. Cells grown anaerobically on various alcohols effectively oxidized methanol in the presence of flavins, cofactor FAD and the methanogenic cofactor F₄₂₀, suggesting a constitutive alcohol oxidizing capacity. In cells grown anaerobically on methanol, the rate of methanol oxidation with F₄₂₀ was three times that of FAD. Efficient utilization of alcohols in the presence of F₄₂₀ is a novel feature of the present study. The results suggest that utilization of methanol by the mixed culture would involve metabolic interactions between thePseudomonas sp. and the methanogen(s). Methylotrophic, methanogenic partnership involving an aerobe is a novel feature hitherto unreported among anaerobic syntrophic associations and is of ecological significance.     

Detection of Metabolic Key Enzymes of Methane Turnover Processes in Cold Seep Microbial Biofilms

In anoxic environments, methane oxidation is conducted in a syntrophic process between methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB). Microbial mats consisting of ANME, SRB and other microorganisms form methane seep-related carbonate buildups in the anoxic bottom waters of the Black Sea Crimean shelf. To shed light on the localization of the biochemical processes at the level of single cells in the Black Sea microbial mats, we applied antibody-based markers for key enzymes of the relevant metabolic pathways. The dissimilatory adenosine-5′-phosphosulfate (APS) reductase, methyl-coenzyme M reductase (MCR) and methanol dehydrogenase (MDH) were selected to localize sulfate respiration, reverse methanogenesis and aerobic methane oxidation, respectively. The key enzymes could be localized by double immunofluorescence and immunocytochemistry at light- and electron microscopic levels. In this study we show that sulfate reduction is conducted synchronized and in direct proximity to reverse methanogenesis of ANME archaea. Microcolonies in interspaces between ANME/SRB express methanol dehydrogenase, which is indicative for oxidation of C₁ compounds by methylotrophic or methanotrophic bacteria. Thus, in addition to syntrophic AOM, oxygen-dependent processes are also conducted by a small proportion of the microbial population.