Biocatalysis of CO2 and CH4: Key enzymes and challenges.

Mitigating greenhouse gas emissions is a critical challenge for promoting global sustainability. The utilization of CO2 and CH4 as substrates for the production of valuable products offers a promising avenue for establishing an eco-friendly economy. Biocatalysis, a sustainable process utilizing enzymes to facilitate biochemical reactions, plays a significant role in upcycling greenhouse gases. This review provides a comprehensive overview of the enzymes and associated reactions involved in the biocatalytic conversion of CO2 and CH4. Furthermore, the challenges facing the field are discussed, paving the way for future research directions focused on developing robust enzymes and systems for the efficient fixation of CO2 and CH4.

The combined effects of polystyrene nanoplastics with nickel on oxidative stress and related toxic effects to earthworms from individual and cellular perspectives.

Nanoplastics may adsorb other pollutants in the environment due to their high specific surface area and small size. We used earthworms as experimental organisms to evaluate the ecotoxicity of NPs and Ni combined pollution at the individual and cellular levels. The results showed that when only 20 mg/L Ni2+ was added to the combined pollution system, the antioxidant system of earthworm coelomocytes was destroyed to a certain extent, the ROS level increased, the cell viability decreased significantly, and the redox balance was destroyed. With the introduction of PS-NPs and the increase of concentration, the oxidative damage in the coelomocytes of earthworms gradually increased, and finally tended to be stable when the maximum concentration of 50 mg/L PS-NPs and Ni were exposed together. At the animal level, the activities of CAT and SOD decreased within 28 days of exposure, and the combined pollution showed a synergistic effect. At the same time, it promoted the synthesis of GST in earthworms, improved their detoxification ability and reduced oxidative damage. The changes of T-AOC and MDA showed that the combined pollution caused the accumulation of ROS and caused more serious toxicological effects. With the increase of exposure time, the antioxidant system of earthworms was continuously destroyed, and the oxidative damage was serious, which induced more serious lipid peroxidation and caused the damage of earthworm body wall structure.

New mechanistic insights into halogen-dependent cytotoxic pattern of monohaloacetamide disinfection byproducts.

As highly toxic nitrogenous disinfection byproducts (DBPs), monohaloacetamides (monoHAcAms) generally exhibited a cytotoxic rank order of iodoacetamide ˃ bromoacetamide ˃ chloroacetamide. However, the mechanisms underlying the halogen-dependent cytotoxic pattern remain largely veiled as yet. In this work, oxidative stress/damage levels in monoHAcAm-treated Chinese hamster ovary cells were thoroughly analyzed, and binding interactions between monoHAcAms and antioxidative enzyme Cu/Zn-superoxide dismutase (Cu/Zn-SOD) were investigated by multiple spectroscopic techniques and molecular docking. Upon exposure to monoHAcAms, the intracellular levels of key biomarkers associated with oxidative stress/damage, including reactive oxygen species, malondialdehyde, lactate dehydrogenase, 8-hydroxy-2-deoxyguanosine, cell apoptosis, and G1 cell cycle arrest, were all significantly increased in a dose-response manner with the same halogen-dependent rank order as their cytotoxicity. Moreover, this rank order was also determined to be applicable to the monoHAcAm-induced alterations in the conformation, secondary structure, and activity of Cu/Zn-SOD, the microenvironment surrounding aromatic amino acid residues in Cu/Zn-SOD, as well as the predicted binding energy of SOD-monoHAcAm interactions. Our results revealed that the halogen-dependent cytotoxic pattern of monoHAcAms was attributed to their differential capacity to induce oxidative stress/damage and their interaction with antioxidative enzyme, which contribute to a better understanding of the halogenated DBP-induced toxicological mechanisms.

Molecular mechanisms of nano-sized polystyrene plastics induced cytotoxicity and immunotoxicity in Eisenia fetida.

Nanoplastics (NPs) are currently everywhere and environmental pollution by NPs is a pressing global problem. Nevertheless, until now, few studies have concentrated on the mechanisms and pathways of cytotoxic effects and immune dysfunction of NPs on soil organisms employing a multidimensional strategy. Hence, earthworm immune cells and immunity protein lysozyme (LZM) were selected as specific receptors to uncover the underlying mechanisms of cytotoxicity, genotoxicity, and immunotoxicity resulting from exposure to polystyrene nanoplastics (PS-NPs), and the binding mechanisms of PS-NPs-LZM interaction. Results on cells indicated that when earthworm immune cells were exposed to high-dose PS-NPs, it caused a notable rise in the release of reactive oxygen species (ROS), resulting in oxidative stress. PS-NPs exposure significantly decreased the cell viability of earthworm immune cells, inducing cytotoxicity through ROS-mediated oxidative stress pathway, and oxidative injury effects, including reduced antioxidant defenses, lipid peroxidation, DNA damage, and protein oxidation. Moreover, PS-NPs stress inhibited the intracellular LZM activity in immune cells, resulting in impaired immune function and immunotoxicity by activating the oxidative stress pathway mediated by ROS. The results from molecular studies revealed that PS-NPs binding destroyed the LZM structure and conformation, including secondary structure changes, protein skeleton unfolding/loosening, fluorescence sensitization, microenvironment changes, and particle size changes. Molecular docking suggested that PS-NPs combined with active center of LZM easier and inhibited the protein function more, and formed a hydrophobic interaction with TRP 62, a crucial amino acid residue closely associated with the function and conformation of LZM. This is also responsible for LZM conformational changes and functional inhibition /inactivation. These results of this research offer a fresh outlook on evaluating the detriment of NPs to the immune function of soil organisms using cellular and molecular strategies.

Novel evidence on iodoacetic acid-induced immune protein functional and conformational changes: Focusing on cellular and molecular aspects.

Elevated levels of iodide occur in raw water in certain regions, where iodination disinfection byproducts are formed during chloramine-assisted disinfection of naturally iodide-containing water. Iodoacetic acid (IAA) is one of the typical harmful products. The mechanisms underlying IAA-induced immunotoxicity and its direct effects on biomolecules remained unclear in the past. Cellular, biochemical, and molecular methods were used to investigate the mechanism of IAA-induced immunotoxicity and its binding to lysozyme. In the presence of IAA, the cell viability of coelomocytes was significantly reduced to 70.8 %, as was the intracellular lysozyme activity. Upon binding to IAA, lysozyme underwent structural and conformational changes, causing elongation and unfolding of the protein due to loosening of the backbone and polypeptide chains. IAA effectively quenched the fluorescence of lysozyme and induced a reduction in particle sizes. Molecular docking revealed that the catalytic residue, Glu 35, which is crucial for lysozyme activity, resided within the docking range, suggesting the preferential binding of IAA to the active site of lysozyme. Moreover, electrostatic interaction emerged as the primary driving force behind the interaction between IAA and lysozyme. In conclusion, the structural and conformational changes induced by IAA in lysozyme resulted in impaired immune protein function in coelomocytes, leading to cellular dysfunction.

Compound effect and mechanism of oxidative damage induced by nanoplastics and benzo [a] pyrene.

Nanoplastics and polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in soil environments. In order to objectively evaluate the toxic interaction between polystyrene nanoplastics (PS NPs) and benzo [a] pyrene (BaP), oxidative damage at the level of earthworm cells and biomacromolecules was investigated by experiments combined with molecular dynamics simulation. Studies on cells reveal that PS NPs and BaP had synergistic toxicity when it came to causing oxidative stress. Cellular reactive oxygen species (ROS) levels under combined pollutant exposure were 24% and 19% higher, respectively than when PS NPs and BaP were exposed alone (compared to the blank group). In addition, BaP and PS NPs inhibited the ability of CAT to decompose H2O2 by affecting the structure of the proximal amino acid Tyr 357 in the active center of CAT, which exacerbated oxidative stress to a certain extent. Therefore, the synergistic toxic effect of BaP and PS NPs is due to the mutual complement of the two to the induction of protein structural looseness, and the strengthening of the stability of the conjugate (CAT-BaP-PS) under the weak interaction. This work provides a new perspective and approach on how to talk about the toxicity of combined pollutants.

Environmental relevant concentrations of polystyrene nanoplastics and lead co-exposure triggered cellular cytotoxicity responses and underlying mechanisms in Eisenia fetida.

Heavy metal pollution of soils and the widespread use of plastics have caused environmental problems worldwide. Nanoplastics (NPs) contaminants in water and soil environments can adsorb heavy metals, thereby affecting the bioavailability and toxicity of heavy metals. In this paper, the effect of co-exposure of polystyrene microspheres with 100 nm particle size and lead acetate (Pb) on the Eisenia fetida coelomocytes was investigated. The environmental concentration of NPs used was 0.01 mg/L and the concentration of Pb ranged from 0.01 to 1 mg/L, and the exposed cells were incubated at 298 k for 24 h. Our study demonstrated that exposure of cells to environmental relevant concentrations of NPs did not significantly affect the cytotoxicity of Pb exposure. It was shown that co-exposure induced cellular production of reactive oxygen species (ROS, increased to 134.4 %) disrupted the antioxidant system of earthworm body cavity cells, activated superoxide dismutase and catalase (CAT), produced reduced glutathione, and inhibited glutathione-dependent enzyme (GST) activity (Reduced to 64 %). Total antioxidant capacity (T-AOC) is first enhanced against ROS due to the stress of NPs and Pb. When the antioxidant reserves of cells are exhausted, the antioxidant capacity will decrease. The level of malondialdehyde, a biomarker of eventual lipid peroxidation, increased to 231.7 %. At the molecular level, due to co-exposure to NPs and Pb, CAT was loosely structured and the secondary structure is misfolded, which was responsible for exacerbating oxidative damage in E. fetida coelomocytes. The findings of this study have significant implications for the toxicological interaction and future risk assessment of co-contamination of NPs and Pb in the environment.

Decoding the biological toxicity of phenanthrene on intestinal cells of Eisenia fetida: Effects, toxicity pathways and corresponding mechanisms.

Phenanthrene is frequently detected and exists extensively in the soil environment, and its residues inevitably impose a significant threat to soil organisms. Exposure to and toxicity of phenanthrene on earthworms has been extensively studied before, however, the possible mechanisms and related pathways associated with phenanthrene-triggered toxicity at the intestinal cell level remain unclear. Herein, primary intestinal cells isolated from Eisenia fetida (Annelida, Oligochaeta) intestine were used as targeted receptors to probe the molecular mechanisms involved in ROS-mediated damaging effects and the potential pathways of phenanthrene-induced toxicity at cellular and sub-cellular levels. Results indicated that phenanthrene exposure induced oxidative stress by activating intracellular ROS (elevated O2-, H2O2, and OH- content) bursts in E. fetida intestinal cells, causing various oxidative damage effects, including lipid peroxidation (increased MDA content), protein oxidation (enhanced PCO levels), and DNA damage (enhanced 8-OHdG levels). The enzymatic and non-enzymatic strategies in earthworm cells were activated to mitigate these detrimental effects by regulating ROS-mediated pathways involving defense regulation. Also, phenanthrene stress destroyed the cell membrane of E. fetida intestinal cells, resulting in cellular calcium homeostasis disruption and cellular energetic alteration, ultimately causing cytotoxicity and cell apoptosis/death. More importantly, the mitochondrial dysfunction in E. fetida cells was induced by phenanthrene-caused mitochondrial membrane depolarization, which in turn caused un-controlled ROS burst and induced apoptosis through mitochondria-mediated caspase-3 activation and ROS-mediated mitochondrial-dependent pathway. Furthermore, exposure to phenanthrene activated an abnormal mRNA expression profile associated with defense regulation (e.g., Hsp70, MT, CRT, SOD, CAT, and GST genes) in E. fetida intestinal cells, resulting in various cellular dysfunctions and pathological conditions, eventually, apoptotic cell death. Taken together, this study offers valuable insights for probing the toxic effects and underlying mechanisms posed by phenanthrene at the intestinal cell level, and is of great significance to estimate the detrimental side effects of phenanthrene on soil ecological health.

Mechanistic insights into pyridine exposure induced toxicity in model Eisenia fetida species: Evidence from whole-animal, cellular, and molecular-based perspectives.

Pyridine and its derivatives are widely used in many applications and inevitably cause extreme scenarios of serious soil contamination, which pose a threat to soil organisms. Still, the eco-toxicological effects and underlying mechanisms of pyridine-caused toxicity toward soil fauna have not been well established. Thus, earthworms (Eisenia fetida), coelomocytes, and oxidative stress-related proteins were selected as targeted receptors to probe the ecotoxicity mechanism of extreme pyridine soil exposure targeted to earthworms by using a combination of in vivo animal experiments, cell-based in vitro tests, in vitro functional and conformational analyses, and in silico analyses. The results showed that pyridine caused severe toxicity to E. fetida at extreme environmental concentrations. Exposure of pyridine induced excessive ROS formation in earthworms, causing oxidative stress and various deleterious effects, including lipid damage, DNA injury, histopathological change, and decreased defense capacity. Also, pyridine destroyed the cell membrane of earthworm coelomic cells and triggered a significant cytotoxicity. Importantly, the intracellular ROS (e.g., O2-, H2O2, and OH·-) was release-activated, which eventually inducing oxidative stress effects (lipid peroxidation, inhibited defense capacity, and genotoxicity) through the ROS-mediated mitochondrial pathway. Moreover, the antioxidant defence mechanisms in coelomocytes responded quickly to reduce ROS-mediated oxidative injury. It was conformed that the abnormal expression of targeted genes associated with oxidative stress in coelomic cells was activated after pyridine exposure. Particularly, we found that the normal conformation (particle sizes, intrinsic fluorescence, and polypeptide backbone structure) of CAT/SOD was destroyed by the direct binding of pyridine. Furthermore, pyridine bound easily to the active center of CAT, but preferentially to the junction cavity of two subunits of SOD, which is considered to be a reason for impaired protein function in cells and in vitro. Based on these evidences, the ecotoxicity mechanisms of pyridine toward soil fauna are elucidated based on multi-level evaluation.

Metabolic Degradation and Bioactive Derivative Synthesis of Phenazine-1-Carboxylic Acid by Genetically Engineered Pseudomonas chlororaphis HT66.

Phenazine-1-carboxylic acid (PCA) secreted by Pseudomonas chlororaphis has been commercialized and widely employed as an antifungal pesticide. However, it displays potential hazards to nontarget microorganisms and the environment. Although the PCA degradation characteristics have received extensive attention, the biodegradation efficiency is still insufficient to address the environmental risks. In this study, an engineered Pseudomonas capable of degrading PCA was constructed by introducing heterologous PCA 1,2-dioxygenase (PcaA1A2A3A4). By integrating the PCA degradation module in the chemical mutagenesis mutant P3, 7.94 g/L PCA can be degraded in 60 h, which exhibited the highest PCA degradation efficiency to date and was 35.4-fold higher than that of the PCA natural degraders. Additionally, PCA was converted to 1-methoxyphenazine through structure modification by introducing the functional enzymes PhzSPa and PhzMLa, which has good antifungal activity and environmental compatibility. This work demonstrates new possibilities for developing PCA-derived biopesticides and enables targeted control of the impact of PCA in diverse environments.

Palladium-Catalyzed Tandem Nucleophilic Addition/C-H Functionalization of Anilines and Bromoalkynes for the Synthesis of 2-Phenylindoles.

A novel and efficient palladium-catalyzed annulation of anilines with bromoalkynes for the synthesis of 2-phenylindoles has been described. This approach features excellent regio- and stereoselectivities and good functional group tolerance. Preliminary mechanistic studies indicate that anilines undergo anti-nucleophilic addition to bromoalkynes to generate (Z)-N-(2-bromo-1-phenylvinyl) anilines, followed by sequential C-H functionalization to deliver different substituted 2-phenylindoles. This method provides potential applications for the construction of various biologically active compounds.

Potential toxicity of bisphenol A to α-chymotrypsin and the corresponding mechanisms of their binding.

Bisphenol A (BPA) is an endocrine disruptor widely existing in plastics and resins, which can accumulate in animals and human bodies, posing a potential threat to the physiological and biochemical reactions of human beings or other organisms. α-Chymotrypsin is a kind of proteolytic enzyme existing in humans and animals, which can cause diseases when its activity is excessive. However, there is a lack of research on the mechanism of endocrine disruptors affecting α-chymotrypsin activity. In this study, the interaction between BPA and α-chymotrypsin was proved via multiple spectroscopic approaches, enzyme activity change, isothermal titration calorimetry and molecular docking. Results showed that α-chymotrypsin's polypeptide chains were unfolded, and protein skeletons were loosened with the exposure to BPA. α-Helix content increased and ß-sheet content was decreased. The particle size of the BPA-α-chymotrypsin complex became smaller. Fluorescence sensitization may also be explained by a perturbation of the chromophore Trp 141. The thermodynamic parameters of the binding reaction were measured by isothermal titration calorimetry (ITC), which showed that there was hydrophobic interaction between BPA and α-chymotrypsin, which was consistent with the results of molecular docking. Moreover, BPA may stop near the active center of α-chymotrypsin and interact with the key residues His 57 and Ser 195. The above phenomenon explained the result that the activity of α-chymotrypsin increased to 139% when exposed to high dose BPA (40 µM). Taken together, the effects of BPA on the structure and function of α-chymotrypsin were clarified at the molecular level, which made up the gap in the mechanism of BPA on the proteolytic enzyme, and provided a reliable basis for disease avoidance and prevention.

[Electron supply strategies for microbial carbon fixation: a review].

Given the rapid development of technologies in biochemical engineering and genetic engineering, biological capture, conversion and utilization of greenhouse gases (carbon dioxide and methane) into value-added products have been progressed rapidly. The efficiency of electron transfer and energy supply are essential for microbial carbon fixation. In this review, the concepts of direct and indirect electron transfer chains in methanotrophic and chemoautotrophic microbes were introduced firstly. Subsequently, the strategies of supplying light and electrical energy as well as their effects on metabolic flux, synthetic pathway and energy supply efficiency during microbial carbon fixation were discussed. Finally, solutions and application prospects to address the key technical challenges of microbial carbon fixation were discussed.

Production of Antibacterial Questiomycin A in Metabolically Engineered Pseudomonas chlororaphis HT66.

Pseudomonas chlororaphis has been demonstrated as a valuable source of antimicrobial metabolites for plant disease biocontrol and biopesticide development. Although phenazine-1-carboxylic acid (PCA) secreted by P. chlororaphis has been commercialized as an antifungal biopesticide, it shows poor antibacterial activity. Questiomycin A, with versatile antibacterial activities, is mainly discovered in some well-known phenazine-producing strains but not in Pseudomonas. Its low titer hinders practical applications. In this work, a metabolite was first identified as Questiomycin A in P. chlororaphis-derived strain HT66ΔphzBΔNat. Subsequently, Questiomycin A has been elucidated to share the same biosynthesis process with PCA by gene deletion and in vitro assays. Through rational metabolic engineering, heterologous phenoxazinone synthase introduction, and medium optimization, the titer reached 589.78 mg/L in P. chlororaphis, the highest production reported to date. This work contributes to a better understanding of Questiomycin A biosynthesis and demonstrates a promising approach to developing a new antibacterial biopesticide in Pseudomonas.

Transcriptomic profiling of nitrogen fixation and the role of NifA in Methylomicrobium buryatense 5GB1.

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.

Bio-valorization of C1 gaseous substrates into bioalcohols: Potentials and challenges in reducing carbon emissions.

C1 gaseous substrates (CH4, CO2, and CO) derived from natural gas, biogas, and syngas, are of interest due to their threats to the environment or inefficient utilization. Benefiting from advanced genetic editing tools and bioconversion strategies, metabolically engineered C1-gas-utilizing microorganisms (CGUM), such as methanotrophs, cyanobacteria, and acetogens, are capable of utilizing C1 gaseous feedstocks as the sole substrates for cell growth and synthesis of chemicals and biofuels. In this paper, we critically review metabolic pathways related to the assimilation of C1 gaseous substrates for alcohol biosynthesis in several model CGUM. Metabolic engineering approaches utilized to enhance the carbon conversion efficiency, microbial growth and biosynthesis of desired alcohols are summarized, including the regulation of C1 gaseous substrates activation and electron and energy supply, the accumulation of key intermediates, and the manipulation of target gene expression to optimize carbon flux to bioalcohols. In addition, challenges in the efficient microbial conversion of C1 gaseous substrates are explored and discussed. The strategies of bioalcohol biosynthesis presented here could guide the development of a variety of efficient biological routes for CH4, CO2, and CO utilization in the future.

Systems Metabolic Engineering of Methanotrophic Bacteria for Biological Conversion of Methane to Value-Added Compounds.

Methane is considered the carbon source with the highest potential in industrial biotechnology because of its abundance and sustainability. The biological conversion of methane into chemicals or fuels can not only reduce greenhouse gas emissions, but can also substitute edible substrates used in biorefineries. Methanotrophs that can utilize methane as the sole energy and carbon source play a significant role in the ecology of methane. Studies on metabolically-engineered methanotrophs have attracted extensive attention in recent years. In this chapter, the approaches and strategies of methanotrophic cell factory construction are summarized based on synthetic biology tools, systematic manipulation, metabolic modeling, and carbon flux enhancement. Finally, the challenges and opportunities for methane bioconversion by methanotrophs are discussed based on industrial applications.

Unraveling the biosynthesis of penicillenols by genome mining PKS-NRPS gene clusters in Penicillium citrinum.

Penicillenols belong to the family of tetramic acids with anticancer and antibacterial activities. Here, we report the discovery of the biosynthetic gene cluster (pnc) for penicillenol A1 and E in Penicillium citrinum ATCC9849 by genome mining. We discover the pnc cluster based on the results of gene deletions in P. citrinum and gene cluster heterologous expression in Aspergillus nidulans. We also propose the assembly line of the PKS module in PncA with the reduction by PncB provides a highly reduce polyketide chain to be further linked with an L-threonine molecule and released from PncA to produce penicillenol E. Further formation of penicillenol A1 requires the N-methylation of tetramic acid group by PncC. Our work deepens the understanding of the biosynthetic logic for N-methylated tetramic acids and contributes to the discovery of new penicillenols by genome mining.

Transcription regulation strategies in methylotrophs: progress and challenges.

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.

Exploration of an Efficient Electroporation System for Heterologous Gene Expression in the Genome of Methanotroph.

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.