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| Framework of the heterologous expression of 2-ketoisovalerate decarboxylase (KivD) in strain Methylomicrobium buryatense 5GB1S. (A) Artificial pathway for isobutyraldehyde production from methanol or methane in strain M. buryatense 5GB1S. (B) Scheme of the plasmid-based expression of the foreign gene kivd in strain M. buryatense 5GB1S. (C) Scheme of the integration of the foreign gene kivd into the chromosome of strain M. buryatense 5GB1S. Word in red represents an exogenous gene. Words in black represent endogenous genes. These genes coded for acetohydroxyacid synthase (ilvHI), acetohydroxyacid isomeroreductase (ilvC), and dihydroxyacid dehydratase (ilvD). Intersecting lines indicate homologous recombination. LF, left flanking region; RF, right flanking region.
Source publication
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 a...
Contexts in source publication
Context 1
... evaluate the SSCE method, M. buryatense 5GBlS was engineered for the biosynthesis of isobutyraldehyde, which is the key precursor of isobutanol applied as an attractive fuel substitute ( Atsumi et al., 2010). The gene encoding alpha-ketoisovalerate decarboxylase (KivD) from Lactococcus lactis, which can catalyze 3-methyl-2-oxybutyric acid to isobutyraldehyde, was used as the heterologous gene, as shown in Figure 2A. Two strategies can be employed for the expression of the heterologous gene kivd in methanotrophs: a plasmid-based expression ( Figure 2B) and a chromosome-based homologous recombination (Figure 2C). ...
Context 2
... gene encoding alpha-ketoisovalerate decarboxylase (KivD) from Lactococcus lactis, which can catalyze 3-methyl-2-oxybutyric acid to isobutyraldehyde, was used as the heterologous gene, as shown in Figure 2A. Two strategies can be employed for the expression of the heterologous gene kivd in methanotrophs: a plasmid-based expression ( Figure 2B) and a chromosome-based homologous recombination (Figure 2C). Although the former has been applied in many model strains with better outcomes, the R-M system in methanotrophs results in a lower efficiency. ...
Context 3
... gene encoding alpha-ketoisovalerate decarboxylase (KivD) from Lactococcus lactis, which can catalyze 3-methyl-2-oxybutyric acid to isobutyraldehyde, was used as the heterologous gene, as shown in Figure 2A. Two strategies can be employed for the expression of the heterologous gene kivd in methanotrophs: a plasmid-based expression ( Figure 2B) and a chromosome-based homologous recombination (Figure 2C). Although the former has been applied in many model strains with better outcomes, the R-M system in methanotrophs results in a lower efficiency. ...
Context 4
... a modified chromosome-based method was developed to provide a fast and efficient system. As shown in Figure 3 and Supplementary Figure 2, the recombinant strain (M. buryatense 5GBlSfadE:Km r :kivd) was constructed by replacing the specific site of the fadE gene in the genome with the exogenous DNA fragment of kivd and Km r . ...
Citations
... All these construction methods are followed by transformation into bacteria. While chemical transformation methods such as PEG (polyethylene glycol) mediated transformation can be used, electroporation-mediated transformation has been preferred due to higher efficiency [98]. Electroporation is relatively easy for direct and high-efficiency insertion of DNA fragments into genome-specific sites when compared with other conjugation-based transformation methods [98]. ...
... While chemical transformation methods such as PEG (polyethylene glycol) mediated transformation can be used, electroporation-mediated transformation has been preferred due to higher efficiency [98]. Electroporation is relatively easy for direct and high-efficiency insertion of DNA fragments into genome-specific sites when compared with other conjugation-based transformation methods [98]. Depending on the strategy chosen during the design step, genome editing in bacteria is performed either by CRISPR-Cas, CRISPRi, CRISPRa, base editing, or prime editing. ...
Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas system has been the focus since its first introduction to genome editing research. It acts as an adaptive immune system in many prokaryotes by detecting and degrading viral/invasive DNA/RNA repurposed for targeted genome editing in various organisms, including bacterial species relevant to industrial biotechnology. Targeted genome editing in bacteria has been implemented for multiple purposes, such as method development, understanding knowledge of metabolic processes and improving the productivity of industrial products. With the increasing share of biotechnological products, bacteria have been the subject in the field of genome editing. This review summarizes the immunological mechanisms of CRISPR-Cas systems, the variation in classification of CRISPR-Cas systems by highlighting their features and the current applications of CRISPR-based genome editing in industrially relevant bacteria. In addition, an extensive literature review focused on recent advances has been presented in bacterial species according to industrial significance.
... To increase the efficiency of electroporation in methanotrophs that possess both pMMO and sMMO, they are grown on a mineral medium with a low CuSO 4 content (<10 μM), which determines the decreased ICM level (Ro and Rosenzweig, 2018;. In a recent study, Hu et al. (2021) improved the efficiency of electroporation by optimizing several key parameters, in particular, preparation of competent cells, electroporation conditions, recovery period, and antibiotic concentration. Using an approach based on homologous recombination and efficient transformation, targeted gene deletion and expression of a heterologous gene were both achieved at the same time by means of direct electroporation with linear DNA fragments obtained by PCR. ...
... Using an approach based on homologous recombination and efficient transformation, targeted gene deletion and expression of a heterologous gene were both achieved at the same time by means of direct electroporation with linear DNA fragments obtained by PCR. In this way, the authors were able to construct an isobutyraldehyde producer strain based on Mm. buryatense (Hu et al., 2021). ...
... More than 900 mg/L isobutanol has been demonstrated by using CO 2 as the sole carbon source in the autotrophic cultivation of Synechocystis PCC 6803 [18]. In recent years, native and/or non-native C1-utilizing microbes have been modified or constructed by using genetic engineering tools, systematic manipulation, metabolic modeling, and carbon flux simulation to improve the C1-gas utilization rate [19,20]. However, the challenges and opportunities for methane bioconversion into isobutanol by methanotrophs remain in both scientific and industrial applications. ...
Background
The dramatic increase in greenhouse gas (GHG) emissions, which causes serious global environmental issues and severe climate changes, has become a global problem of concern in recent decades. Currently, native and/or non-native C1-utilizing microbes have been modified to be able to effectively convert C1-gases (biogas, natural gas, and CO2) into isobutanol via biological routes. Even though the current experimental results are satisfactory in lab-scale research, the techno-economic feasibility of C1 gas-derived isobutanol production at the industrial scale still needs to be analyzed and evaluated, which will be essential for the future industrialization of C1-gas bioconversion. Therefore, techno-economic analyses were conducted in this study with comparisons of capital cost (CAPEX), operating cost (OPEX), and minimum isobutanol selling price (MISP) derived from biogas (scenario #1), natural gas (scenario #2), and CO2 (scenario #3) with systematic economic assessment.
Results
By calculating capital investments and necessary expenses, the highest CAPEX (67 MM) were projected in scenario #1 and scenario #2, respectively. Because of the lower CAPEX and OPEX from scenario #3, the results revealed that bioconversion of CO2 into isobutanol temporally exhibited the best economic performance with an MISP of 0.99/kg in a long-term case study.
Conclusions
This study provides a comprehensive assessment of the bioconversion of C1-gases into isobutanol in terms of the bioprocess design, mass/energy calculation, capital investment, operating expense, sensitivity analysis, and minimum selling price. Compared with isobutanol derived from biogas and natural gas, the CO2-based isobutanol showed better economic feasibility. A market competitive isobutanol derived from CO2 is predicable with lower CO2 cost, better isobutanol titer, and higher annual capacity. This study will help researchers and decision-makers explore innovative and effective approaches to neutralizing GHGs and focus on key economic-driving forces to improve techno-economic performance.
... Thus, the development of efficient electroporation methods to replace the currently used conjugation methods is essential. In most recent studies, electroporation efficiency was improved by optimization of several key parameters such as competent cell preparation, electroporation condition, recovery time, and antibiotic concentration maximum, resulting in an efficiency of 719 ± 22.5 CFU/µg DNA [27]. By using the novel electroporation method, the engineered M. buryatense 5GB1, in which a heterologous kivD replaced the endogenous fadE, produced isobutyraldehyde with a titer of 3.3 mg/L. ...
Methane and carbon dioxide (CO2) account for the most abundant greenhouse gases (GHGs). The removal of atmospheric methane and CO2 is an urgent mission contributing to the minimization of climate change. In this context, the methylotrophic bacteria-based technologies capturing and assimilating methane, CO2 and methane/CO2 derivatives, such as methanol and formate, are promising to reduce atmospheric methane and CO2. Herein, we emphasized the outstanding of methylotrophs in bioconversion of methane, CO2, methanol and formate, which are regarded as one-carbon (C1) substrates. Innovative strategies including co-substrate cultivation, power-generation reinforcement, and rational metabolic engineering for improving the efficiency of C1 bioconversion are highlighted. Additionally, we updated the advances in genetic tools to manipulate methylotrophs and discussed the possible applications of methylotrophs in sustainable agriculture. We believe that upgrading methylotrophic hosts towards efficient C1-conversion is a sustainable approach to achieving C1-based industrial manufacturing.
... To obtain a nifA gene deficiency mutant of M. buryatense 5GB1, the linear fragment containing a kanamycin-resistant cassette was prepared as the previous report (Hu et al. 2021;Liu et al. 2020). The mutant strain M. buryatense 5GB1ΔnifA was constructed via homologous recombination of a linear fragment. ...
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.
