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Overview of the differential expression of genes encoding enzymes involving in nitrogen fixation, methane metabolism, ClpX system, and Pst system in M. buryatense 5GB1 in condition #3 (0.93) compared with #2 (0.58), #4 (1.31), and #5 (5.24). Dotted arrow indicates multi-step reaction, while solid arrow represents one-step reaction. The numbers in shadow at right-side of protein represent the log2-based changes of differentially expressed gene under condition #3 compared to #2, #4, or #5, respectively. The red shadow means upregulation, while the blue shadow means downregulation.
Source publication
The methane (CH4)/oxygen (O2) gas supply ratios significantly affect the cell growth and metabolic pathways of aerobic obligate methanotrophs. However, few studies have explored the CH4/O2 ratios of the inlet gas, especially for the CH4 concentrations within the explosion range (5∼15% of CH4 in air). This study thoroughly investigated the molecular...
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The development of biorefineries for a sustainable bioeconomy has been driven by the concept of utilizing environmentally friendly and cost-effective renewable energy sources. Methanotrophic bacteria with a unique capacity to utilize methane as a carbon and energy source can serve as outstanding biocatalysts to develop C1 bioconversion technology....
Citations
... Methane serves as both the carbon and energy source for methanotrophs, and oxygen acts as a critical electron acceptor. Maintaining a balanced ratio of carbon and oxygen is essential for their optimal growth [28,29]. Methane monooxygenase (MMO), existing in both soluble (sMMO) and particulate (pMMO) forms, is regulated by copper ions (Cu 2+ ). ...
Methanotrophs hold significant potential in global methane mitigation and resource recovery. However, the limited rate of cell proliferation remains a significant constraint for large-scale applications. Therefore, screening efficient methanotrophic strains that are suitable for industrial applications to mitigate methane and exploring potential methane resource utilization pathways are of great importance for sustainable development. Gradient dilution and the streak plate method were employed to isolate methanotrophic strains from a previously domesticated methane-oxidizing microbial consortium. We isolated a highly efficient strain, M6, which exhibited a 230% increase in growth rate compared to the laboratory model strain Methylocystis bryophila (M. bryophila). Taxonomic analysis revealed that strain M6 is classified as Methylocystis parvus. Genomic data indicated a diverse range of metabolic functions. In addition to utilizing methane, strain M6 can also utilize citrate to generate energy and intermediate products, addressing issues related to insufficient methane supply or low methane mass transfer efficiency. Metabolic adaptability ensures the stability of its application. The optimal cultivation conditions for strain M6 were determined, characterized by mild and easily implementable parameters. Based on the analysis of the genome and metabolic pathways, strain M6 exhibits potential for the synthesis of bioproducts, such as proteins, lipids, and polyhydroxyalkanoates (PHAs), with the fermentation process not requiring cost-intensive carbon sources, making it both economical and sustainable.
... Nevertheless, as DP3 could grow under nitrogen-starvation conditions, this strain was considered capable of fixing nitrogen in air and supplying it for cell growth, but not at sufficient levels for ectoine synthesis. Methylomicrobium is reported to demonstrate the potential to utilize atmospheric nitrogen gas based on identification of the nif gene cluster and MoFe-containing nitrogenase activity [31][32][33][34][35]. ...
Background
Methane is a greenhouse gas with a significant potential to contribute to global warming. The biological conversion of methane to ectoine using methanotrophs represents an environmentally and economically beneficial technology, combining the reduction of methane that would otherwise be combusted and released into the atmosphere with the production of value-added products.
Results
In this study, high ectoine production was achieved using genetically engineered Methylomicrobium alcaliphilum 20Z, a methanotrophic ectoine-producing bacterium, by knocking out doeA, which encodes a putative ectoine hydrolase, resulting in complete inhibition of ectoine degradation. Ectoine was confirmed to be degraded by doeA to N-α-acetyl-L-2,4-diaminobutyrate under nitrogen depletion conditions. Optimal copper and nitrogen concentrations enhanced biomass and ectoine production, respectively. Under optimal fed-batch fermentation conditions, ectoine production proportionate with biomass production was achieved, resulting in 1.0 g/L of ectoine with 16 g/L of biomass. Upon applying a hyperosmotic shock after high–cell–density culture, 1.5 g/L of ectoine was obtained without further cell growth from methane.
Conclusions
This study suggests the optimization of a method for the high production of ectoine from methane by preventing ectoine degradation. To our knowledge, the final titer of ectoine obtained by M. alcaliphilum 20ZDP3 was the highest in the ectoine production from methane to date. This is the first study to propose ectoine production from methane applying high cell density culture by preventing ectoine degradation.
... Furthermore, there are not any context-specific GSM models constructed for methanotrophs to the best of our knowledge. However, a large number of transcriptomic datasets has been accumulated for diverse methanotrophs that are suitable for further integration into previously developed GSM models (see review in [10,52,[136][137][138][139][140][141][142][143][144][145][146][147]). Consequently, the application of this advanced approach in constraint-based modeling of methanotrophic metabolism could pave additional ways to novel and potentially transformative solutions for C1-biotechnology in order to improve the competitiveness of methane-consuming bacteria as microbial producers. ...
Methanotrophy is the ability of an organism to capture and utilize the greenhouse gas, methane, as a source of energy-rich carbon. Over the years, significant progress has been made in understanding of mechanisms for methane utilization, mostly in bacterial systems, including the key metabolic pathways, regulation and the impact of various factors (iron, copper, calcium, lanthanum, and tungsten) on cell growth and methane bioconversion. The implementation of -omics approaches provided vast amount of heterogeneous data that require the adaptation or development of computational tools for a system-wide interrogative analysis of methanotrophy. The genome-scale mathematical modeling of its metabolism has been envisioned as one of the most productive strategies for the integration of muti-scale data to better understand methane metabolism and enable its biotechnological implementation. Herein, we provide an overview of various computational strategies implemented for methanotrophic systems. We highlight functional capabilities as well as limitations of the most popular web resources for the reconstruction, modification and optimization of the genome-scale metabolic models for methane-utilizing bacteria.
... Besides, some TFs that are not directly related to the target metabolic process can also be reflected from the transcription level due to the interconnected metabolic networks in methylotrophs. Phosphate transport regulatory cluster phoBU in M. buryatense 5GB1 was found to respond to the methane/oxygen ratio in the headspace, confirming an interaction between phosphate transport and carbon fixation (Hu et al. 2020). However, there are still some housekeeping TFs existing in methylotrophs that are unable to exhibit the obvious transcription undulation regardless of the growth conditions. ...
As a promising industrial microorganism, methylotroph is capable of using methane or methanol as the sole carbon source natively, which has been utilized in the biosynthesis of various bioproducts. However, the relatively low efficiency of carbon conversion has become a limiting factor throughout the development of methanotrophic cell factories due to the unclear genetic background. To better highlight their advantages in methane or methanol-based biomanufacturing, some metabolic engineering strategies, including upstream transcription regulation projects, are being popularized in methylotrophs. In this review, several strategies of transcription regulations applied in methylotrophs are summarized and their applications are discussed and prospected.
... Currently, the physiological function and metabolic mechanism of converting carbon sources in methanotrophs have been elucidated to facilitate the biosynthesis of numerous C1-based products Nguyen and Lee 2021). Besides the metabolism of C1-substrates, several methanotrophs have been reported to be able to utilize atmospheric nitrogen gas (N 2 ) due to the confirmation of nif gene cluster, MoFeprotein nitrogenase activity, and related phylogenetic analyses (Dekas et al. 2009;Hu et al. 2020;Jung et al. 2020). Most of the aforementioned works were focused on Shuqi Guo and Tianqing Zhang have contributed equally to this work. ...
... Then, the linear fragment used for gene disruption was obtained by overlap polymerase chain reaction (PCR) of three PCR products. The resulting PCR products were purified using Mon-Pure PCR Clean Kit (Monad Biotech, Wuhan, China) and directly transformed into strain M. buryatense 5GB1 by our optimized electroporation method (Hu et al. 2020). Mutant M. buryatense 5GB1ΔnifA growing on kanamycin (50 μg/ mL) selective plates were further confirmed by PCR using p-nifA-F/R primers and DNA sequencing ( IncP-based broad host range plasmid containing dTomato, Km r to gene nifA being 1527 bp, the PCR product used primers p-nifA-F/R should be 816 bp (the same as the kanamycin gene) in the mutant. ...
... (Shanghai, China). The quality of RNA-Seq fastq data was checked using FastQC program (Hu et al. 2020). Next, data were imported into CLC Genomics Workbench (version 11.0; https:// digit alins ights. ...
Methanotrophs capable of converting C1-based substrates play an important role in the global carbon cycle. As one of the essential macronutrient components in the medium, the uptake of nitrogen sources severely regulates the cell’s metabolism. Although the feasibility of utilizing nitrogen gas (N2) by methanotrophs has been predicted, the mechanism remains unclear. Herein, the regulation of nitrogen fixation by an essential nitrogen-fixing regulator (NifA) was explored based on transcriptomic analyses of Methylomicrobium buryatense 5GB1. A deletion mutant of the nitrogen global regulator NifA was constructed, and the growth of M. buryatense 5GB1ΔnifA exhibited significant growth inhibition compared with wild-type strain after the depletion of nitrate source in the medium. Our transcriptome analyses elucidated that 22.0% of the genome was affected in expression by NifA in M. buryatense 5GB1. Besides genes associated with nitrogen assimilation such as nitrogenase structural genes, genes related to cofactor biosynthesis, electron transport, and post-transcriptional modification were significantly upregulated in the presence of NifA to enhance N2 fixation; other genes related to carbon metabolism, energy metabolism, membrane transport, and cell motility were strongly modulated by NifA to facilitate cell metabolisms. This study not only lays a comprehensive understanding of the physiological characteristics and nitrogen metabolism of methanotrophs, but also provides a potentially efficient strategy to achieve carbon and nitrogen co-utilization.
Key points
• N2 fixation ability of M. buryatense 5GB1 was demonstrated for the first time in experiments by regulating the supply of N2.
• NifA positively regulates nif-related genes to facilitate the uptake of N2 in M. buryatense 5GB1.
• NifA regulates a broad range of cellular functions beyond nif genes in M. buryatense 5GB1.
... More than 900 mg/L of isobutanol has been demonstrated for the rst time by using CO 2 as the sole carbon source in the autotrophic cultivation of Synechocystis PCC 6803 [16]. In recently years, the approaches and strategies of methanotrophic-based CUCFs are constructed and modi ed by using genetic-engineering tool, systematic manipulation, metabolic modeling, and carbon ux simulation [17][18][19]. However, the challenges and opportunities for methane bioconversion into isobutanol by methanotrophs are still remained in both scienti c and industrial applications. ...
Background
The dramatic increase in emissions of greenhouse gases (GHGs) has led to an irreversible effect on the ecosystem, which in turn caused significant harm to human beings and other species. Exploring innovative and effective approaches to neutralizing GHGs is urgently needed. Considering the advancement of synthetic biology and the bioconversion process, C1-utilizing cell factories (CUCFs) have been modified to be able to effectively convert C1-gases includes biogas, natural gas, and carbon dioxide (CO 2 ) into chemicals or fuels via biological routes, which greatly facilitates the inedible carbon sources used in biomanufacturing, increases the potential value of GHGs and meanwhile reduces the GHG emissions. Process design and resultsEven though the current experimental results are satisfactory in lab-scale research, the evaluation of economic feasibility as well as applications of CUCFs in industrial-scale still need to be analyzed. This study designed three scenarios of CUCFs-based conversion of biogas, natural gas, and CO 2 into isobutanol, the detailed techno-economic analyses of these scenarios were conducted with the comparisons of capital cost, operating cost, and minimum isobutanol selling price (MISP). Results revealed that direct bio-conversion of CO 2 by CUCFs into isobutanol exhibited the best economic performance with a MISP of /kg with using ideal targets. Conclusions
Our findings provide a comprehensive assessment of bio-conversion of C1-gases via CUCFs to isobutanol in terms of the bioprocess design, mass/energy calculation, capital investment, operating expense, sensitivity analysis, and environmental impact. It is expected that this study may lead to the paradigm shift in isobutanol synthesis with C1-gases as substrates.
... Methane, derived from natural gas and biogas, is the second most abundant greenhouse gas whose global warming potential is 25 times more than that of carbon dioxide (Fei et al., 2014;Pariatamby et al., 2015). Excessive methane emissions can not only cause a waste of carbon sources but also endanger the environment by causing the global warming effect (Bjorck et al., 2018;Hu et al., 2020). Thus, it is urgent to seek a potential, green, and sustainable strategy for the efficient utilization of methane. ...
One-carbon (C1) substrates such as methane and methanol have been considered as the next-generation carbon source in industrial biotechnology with the characteristics of low cost, availability, and bioconvertibility. Recently, methanotrophic bacteria naturally capable of converting C1 substrates have drawn attractive attention for their promising applications in C1-based biomanufacturing for the production of chemicals or fuels. Although genetic tools have been explored for metabolically engineered methanotroph construction, there is still a lack of efficient methods for heterologous gene expression in methanotrophs. Here, a rapid and efficient electroporation method with a high transformation efficiency was developed for a robust methanotroph of Methylomicrobium buryatense 5GB1. Based on the homologous recombination and high transformation efficiency, gene deletion and heterologous gene expression can be simultaneously achieved by direct electroporation of PCR-generated linear DNA fragments. In this study, the influence of several key parameters (competent cell preparation, electroporation condition, recovery time, and antibiotic concentration) on the transformation efficiency was investigated for optimum conditions. The maximum electroporation efficiency of 719 ± 22.5 CFU/μg DNA was reached, which presents a 10-fold improvement. By employing this method, an engineered M. buryatense 5GB1 was constructed to biosynthesize isobutyraldehyde by replacing an endogenous fadE gene in the genome with a heterologous kivd gene. This study provides a potential and efficient strategy and method to facilitate the cell factory construction of methanotrophs.
Converting greenhouse gases into valuable products has become a promising approach for achieving a carbon‐neutral economy and sustainable development. However, the conversion efficiency depends on the energy yield of the substrate. In this study, we developed an electro‐biocatalytic system by integrating electrochemical and microbial processes to upcycle CO 2 into a valuable product (ectoine) using renewable energy. This system initiates the electrocatalytic reduction of CO 2 to methane, an energy‐dense molecule, which then serves as an electrofuel to energize the growth of an engineered methanotrophic cell factory for ectoine biosynthesis. The scalability of this system was demonstrated using an array of ten 25 cm ² electrochemical cells equipped with a high‐performance carbon‐supported isolated copper catalyst. The system consistently generated methane at the cathode under a total partial current of approximately −37 A (~175 mmol CH4 h ⁻¹ ) and O 2 at the anode under a total partial current of approximately 62 A (~583 mmol O2 h ⁻¹ ). This output met the requirements of a 3‐L bioreactor, even at maximum CH 4 and O 2 consumption, resulting in the high‐yield conversion of CO 2 to ectoine (1146.9 mg L ⁻¹ ). This work underscores the potential of electrifying the biosynthesis of valuable products from CO 2 , providing a sustainable avenue for biomanufacturing and energy storage.
Converting greenhouse gases into valuable products has become a promising approach for achieving a carbon‐neutral economy and sustainable development. However, the conversion efficiency depends on the energy yield of the substrate. In this study, we developed an electro‐biocatalytic system by integrating electrochemical and microbial processes to upcycle CO2 into a valuable product (ectoine) using renewable energy. This system initiates the electrocatalytic reduction of CO2 to methane, an energy‐dense molecule, which then serves as an electrofuel to energize the growth of an engineered methanotrophic cell factory for ectoine biosynthesis. The scalability of this system was demonstrated using an array of ten 25 cm² electrochemical cells equipped with a high‐performance carbon‐supported isolated copper catalyst. The system consistently generated methane at the cathode under a total partial current of approximately −37 A (~175 mmolCH4 h⁻¹) and O2 at the anode under a total partial current of approximately 62 A (~583 mmolO2 h⁻¹). This output met the requirements of a 3‐L bioreactor, even at maximum CH4 and O2 consumption, resulting in the high‐yield conversion of CO2 to ectoine (1146.9 mg L⁻¹). This work underscores the potential of electrifying the biosynthesis of valuable products from CO2, providing a sustainable avenue for biomanufacturing and energy storage.
