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Since their first industrial application in the acetone-butanol-ethanol (ABE) fermentation in the early 1900s, Clostridia have found large application in biomass biorefining. Overall, their fermentation products include organic acids (e.g., acetate, butyrate, lactate), short chain alcohols (e.g., ethanol, n-butanol, isobutanol), diols (e.g., 1,2-propanediol, 1,3-propanediol) and H2 which have several applications such as fuels, building block chemicals, solvents, food and cosmetic additives. Advantageously, several clostridial strains are able to use cheap feedstocks such as lignocellulosic biomass, food waste, glycerol or C1-gases (CO2, CO) which confer them additional potential as key players for the development of processes less dependent from fossil fuels and with reduced greenhouse gas emissions. The present review aims to provide a survey of research progress aimed at developing Clostridium-mediated biomass fermentation processes, especially as regards strain improvement by metabolic engineering.

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Cyanobacteria are oxygen-evolving prokaryotes that can be engineered for biofuel production from solar energy, CO2, and water. Isobutanol (IB) has the potential to serve as an alternative fuel and important chemical feedstock. The research involves engineering Synechocystis sp. PCC 6803, for photosynthetic isobutanol production via the 2-keto-acid pathway and their cultivation in lab-scale photobioreactors. This synthetic pathway involves the heterologous expression of two enzymes, α-ketoisovalerate decarboxylase (Kivd) and alcohol dehydrogenase (Yqhd), under a strong light-inducible promotor, psbA2, known to show increased gene expression under high light. The use of psbA2 could be a valuable strategy for isobutanol production as economic scaling up demands the utilization of natural sunlight, which also provides very high light intensity at midday, facilitating increased production. The study reports isobutanol production from engineered strains containing both pathway genes and with only kivd. In shake flask studies, the highest isobutanol titre of 75 mg L-1 (12th day) was achieved from an engineered strain DM12 under optimized light intensity. DM12 was cultivated in a 2 L flat panel photobioreactor, resulting in a maximum isobutanol titre of 371.8 mg L-1 (10th day) with 2 % CO2 and 200 µmol photons m-2 s-1. Cultivation of DM12 in a photobioreactor under mimic diurnal sunlight demonstrated the highest productivity of 39 mg L-1 day-1 with the maximum titre of 308.5 mg L-1 (9th day). This work lays the foundation for sustainable, large-scale biobutanol production using solar energy.

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To develop a cost-effective microbial cell factory for the production of biofuels and biochemicals, an understanding of tolerant mechanisms is vital for the construction of robust host strains. Here, we characterized a new function of a key metabolic transcription factor named Znf1 and its involvement in stress response in Saccharomyces cerevisiae to enhance tolerance to advanced biofuel, isobutanol. RNA-sequencing analysis of the wild-type versus the znf1Δ deletion strains in glucose revealed a new role for transcription factor Znf1 in the pentose phosphate pathway (PPP) and energy generation. The gene expression analysis confirmed that isobutanol induces an adaptive cell response, resulting in activation of ATP1-3 and COX6 expression. These genes were Znf1 targets that belong to the electron transport chain, important to produce ATPs. Znf1 also activated PPP genes, required for the generation of key amino acids, cellular metabolites, and maintenance of NADP/NADPH redox balance. In glucose, Znf1 also mediated the upregulation of valine biosynthetic genes of the Ehrlich pathway, namely ILV3, ILV5, and ARO10, associated with the generation of key intermediates for isobutanol production. Using S. cerevisiae knockout collection strains, cells with deleted transcriptional regulatory gene ZNF1 or its targets displayed hypersensitivity to isobutanol and acid inhibitors; in contrast, overexpression of ZNF1 enhanced cell survival. Thus, the transcription factor Znf1 functions in the maintenance of energy homeostasis and redox balance at various checkpoints of yeast metabolic pathways. It ensures the rapid unwiring of gene transcription in response to toxic products/by-products generated during biofuel production. Importantly, we provide a new approach to enhance strain tolerance during the conversion of glucose to biofuels.

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The widespread application of electrochemical hydrogen production faces significant challenges, primarily attributed to the high overpotential of the oxygen evolution reaction (OER) in conventional water electrolysis. To address this issue, an effective strategy involves substituting OER with the value-added oxidation of biomass feedstock, reducing the energy requirements for electrochemical hydrogen production while simultaneously upgrading the biomass. Herein, we introduce an electrocatalytic approach for the value-added oxidation of isobutanol, a high energy density bio-fuel, coupled with hydrogen production. This approach offers a sustainable route to produce the valuable fine chemical isobutyric acid under mild condition. The electrodeposited Ni(OH)2 electrocatalyst exhibits exceptional electrocatalytic activity and durability for the electro-oxidation of isobutanol, achieving an impressive faradaic efficiency of up to 92.4 % for isobutyric acid at 1.45 V vs. RHE. Mechanistic insights reveal that side reactions predominantly stem from the oxidative C-C cleavage of isobutyraldehyde intermediate, forming by-products including formic acid and acetone. Furthermore, we demonstrate the electro-oxidation of isobutanol coupled with hydrogen production in a two-electrode undivided cell, notably reducing the electrolysis voltage by approximately 180 mV at 40 mA cm-2. Overall, this work represents a significant step towards improving the cost-effectiveness of hydrogen production and advancing the conversion of bio-fuels.

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Only trace amount of isobutanol is produced by the native Saccharomyces cerevisiae via degradation of amino acids. Despite several attempts using engineered yeast strains expressing exogenous genes, catabolite repression of glucose must be maintained together with high activity of downstream enzymes, involving iron-sulfur assimilation and isobutanol production. Here, we examined novel roles of nonfermentable carbon transcription factor Znf1 in isobutanol production during xylose utilization. RNA-seq analysis showed that Znf1 activates genes in valine biosynthesis, Ehrlich pathway and iron-sulfur assimilation while coupled deletion or downregulated expression of BUD21 further increased isobutanol biosynthesis from xylose. Overexpression of ZNF1 and xylose-reductase/dehydrogenase (XR-XDH) variants, a xylose-specific sugar transporter, xylulokinase, and enzymes of isobutanol pathway in the engineered S. cerevisiae pho13gre3Δ strain resulted in the superb ZNXISO strain, capable of producing high levels of isobutanol from xylose. The isobutanol titer of 14.809 ± 0.400 g/L was achieved, following addition of 0.05 g/L FeSO4.7H2O in 5 L bioreactor. It corresponded to 155.88 mg/g xylose consumed and + 264.75% improvement in isobutanol yield. This work highlights a new regulatory control of alternative carbon sources by Znf1 on various metabolic pathways. Importantly, we provide a foundational step toward more sustainable production of advanced biofuels from the second most abundant carbon source xylose.

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Due to the limited resources and environmental problems associated with fossil fuels, there is a growing interest in utilizing renewable resources for the production of biofuels through microbial fermentation. Isobutanol is a promising biofuel that could potentially replace gasoline. However, its production efficiency is currently limited by the use of naturally isolated microorganisms. These naturally isolated microorganisms often encounter problems such as a limited range of substrates, low tolerance to solvents or inhibitors, feedback inhibition, and an imbalanced redox state. This makes it difficult to improve their production efficiency through traditional process optimization methods. Fortunately, recent advancements in genetic engineering technologies have made it possible to enhance microbial hosts for the increased production of isobutanol from renewable resources. This review provides a summary of the strategies and synthetic biology approaches that have been employed in the past few years to improve naturally isolated or non-natural microbial hosts for the enhanced production of isobutanol by utilizing different renewable resources. Furthermore, it also discusses the challenges that are faced by engineered microbial hosts and presents future perspectives to enhancing isobutanol production. KEY POINTS: • Promising potential of isobutanol to replace gasoline • Engineering of native and non-native microbial host for isobutanol production • Challenges and opportunities for enhanced isobutanol production.

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As a renewable energy from biomass, isobutanol is considered as a promising alternative to fossil fuels. To biotechnologically produce isobutanol, strain development using industrial microbial hosts, such as Escherichia coli, has been conducted by introducing a heterologous isobutanol synthetic pathway. However, the toxicity of produced isobutanol inhibits cell growth, thereby restricting improvements in isobutanol titer, yield, and productivity. Therefore, the development of robust microbial strains tolerant to isobutanol is required. In this study, isobutanol-tolerant mutants were isolated from two E. coli parental strains, E. coli BL21(DE3) and MG1655(DE3), through adaptive laboratory evolution (ALE) under high isobutanol concentrations. Subsequently, 16 putative genes responsible for isobutanol tolerance were identified by transcriptomic analysis. When overexpressed in E. coli, four genes (fadB, dppC, acs, and csiD) conferred isobutanol tolerance. A fermentation study with a reverse engineered isobutanol-producing E. coli JK209 strain showed that fadB or dppC overexpression improved isobutanol titers by 1.5 times, compared to the control strain. Through coupling adaptive evolution with transcriptomic analysis, new genetic targets utilizable were identified as the basis for the development of an isobutanol-tolerant strain. Thus, these new findings will be helpful not only for a fundamental understanding of microbial isobutanol tolerance but also for facilitating industrially feasible isobutanol production.

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BACKGROUND: The microbial production of isobutanol holds promise to become a sustainable alternative to fossil-based synthesis routes for this important chemical. Escherichia coli has been considered as one production host, however, due to redox imbalance, growth-coupled anaerobic production of isobutanol from glucose in E. coli is only possible if complex media additives or small amounts of oxygen are provided. These strategies have a negative impact on product yield, productivity, reproducibility, and production costs. RESULTS: In this study, we propose a strategy based on acetate as co-substrate for resolving the redox imbalance. We constructed the E. coli background strain SB001 (ΔldhA ΔfrdA ΔpflB) with blocked pathways from glucose to alternative fermentation products but with an enabled pathway for acetate uptake and subsequent conversion to ethanol via acetyl-CoA. This strain, if equipped with the isobutanol production plasmid pIBA4, showed robust exponential growth (µ = 0.05 h-1) under anaerobic conditions in minimal glucose medium supplemented with small amounts of acetate. In small-scale batch cultivations, the strain reached a glucose uptake rate of 4.8 mmol gDW-1 h-1, a titer of 74 mM and 89% of the theoretical maximal isobutanol/glucose yield, while secreting only small amounts of ethanol synthesized from acetate. Furthermore, we show that the strain keeps a high metabolic activity also in a pulsed fed-batch bioreactor cultivation, even if cell growth is impaired by the accumulation of isobutanol in the medium. CONCLUSIONS: This study showcases the beneficial utilization of acetate as a co-substrate and redox sink to facilitate growth-coupled production of isobutanol under anaerobic conditions. This approach holds potential for other applications with different production hosts and/or substrate-product combinations.

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Aiming to overcome the depletion of fossil fuels and serious environmental pollution, biofuels such as isobutanol have garnered increased attention. Among different synthesis methods, the microbial fermentation of isobutanol from raw substrate is a promising strategy due to its low cost and environmentally friendly and optically pure products. As an important component of lignocellulosics and the second most common sugar in nature, xylose has become a promising renewable resource for microbial production. However, bottlenecks in xylose utilization limit its wide application as substrates. In this work, an isobutanol synthetic pathway from xylose was first constructed in E. coli MG1655 through the combination of the Ehrlich and Dahms pathways. The engineering of xylose transport and electron transport chain complexes further improved xylose assimilation and isobutanol production. By optimizing xylose supplement concentration, the recombinant E. coli strain BWL4 could produce 485.35 mg/L isobutanol from 20 g/L of xylose. To our knowledge, this is the first report related to isobutanol production using xylose as a sole carbon source in E. coli. Additionally, a glucose-xylose mixture was utilized as the carbon source. The Entner-Doudorof pathway was used to assimilate glucose, and the Ehrlich pathway was applied for isobutanol production. After carefully engineering the recombinant E. coli, strain BWL9 could produce 528.72 mg/L isobutanol from a mixture of 20 g/L glucose and 10 g/L xylose. The engineering strategies applied in this work provide a useful reference for the microbial production of isobutanol from xylose or glucose-xylose mixture.

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BACKGROUND: Cyanobacteria are emerging as green cell factories for sustainable biofuel and chemical production, due to their photosynthetic ability to use solar energy, carbon dioxide and water in a direct process. The model cyanobacterial strain Synechocystis sp. PCC 6803 has been engineered for the isobutanol and 3-methyl-1-butanol production by introducing a synthetic 2-keto acid pathway. However, the achieved productions still remained low. In the present study, diverse metabolic engineering strategies were implemented in Synechocystis sp. PCC 6803 for further enhanced photosynthetic isobutanol and 3-methyl-1-butanol production. RESULTS: Long-term cultivation was performed on two selected strains resulting in maximum cumulative isobutanol and 3-methyl-1-butanol titers of 1247 mg L-1 and 389 mg L-1, on day 58 and day 48, respectively. Novel Synechocystis strain integrated with a native 2-keto acid pathway was generated and showed a production of 98 mg isobutanol L-1 in short-term screening experiments. Enhanced isobutanol and 3-methyl-1-butanol production was observed when increasing the kivdS286T copy number from three to four. Isobutanol and 3-methyl-1-butanol production was effectively improved when overexpressing selected genes of the central carbon metabolism. Identified genes are potential metabolic engineering targets to further enhance productivity of pyruvate-derived bioproducts in cyanobacteria. CONCLUSIONS: Enhanced isobutanol and 3-methyl-1-butanol production was successfully achieved in Synechocystis sp. PCC 6803 strains through diverse metabolic engineering strategies. The maximum cumulative isobutanol and 3-methyl-1-butanol titers, 1247 mg L-1 and 389 mg L-1, respectively, represent the current highest value reported. The significantly enhanced isobutanol and 3-methyl-1-butanol production in this study further pave the way for an industrial application of photosynthetic cyanobacteria-based biofuel and chemical synthesis from CO2.

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BACKGROUND: With concerns about depletion of fossil fuel and environmental pollution, synthesis of biofuels such as isobutanol from low-cost substrate by microbial cell factories has attracted more and more attention. As one of the most promising carbon sources instead of food resources, acetate can be utilized by versatile microbes and converted into numerous valuable chemicals. RESULTS: An isobutanol synthetic pathway using acetate as sole carbon source was constructed in E. coli. Pyruvate was designed to be generated via acetyl-CoA by pyruvate-ferredoxin oxidoreductase YdbK or anaplerotic pathway. Overexpression of transhydrogenase and NAD kinase increased the isobutanol titer of recombinant E. coli from 121.21 mg/L to 131.5 mg/L under batch cultivation. Further optimization of acetate supplement concentration achieved 157.05 mg/L isobutanol accumulation in WY002, representing the highest isobutanol titer by using acetate as sole carbon source. CONCLUSIONS: The utilization of acetate as carbon source for microbial production of valuable chemicals such as isobutanol could reduce the consumption of food-based substrates and save production cost. Engineering strategies applied in this study will provide a useful reference for microbial production of pyruvate derived chemical compounds from acetate.

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Consolidated bioprocessing (CBP) of lignocellulosic biomass uses cellulolytic microorganisms to enable enzyme production, saccharification, and fermentation to produce biofuels, biochemicals, and biomaterials in a single step. However, understanding and redirecting metabolisms of these microorganisms compatible with CBP are limited. Here, a cellulolytic thermophile Clostridium thermocellum was engineered and demonstrated to be compatible with CBP integrated with a Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment for conversion of hardwood poplar into short-chain esters with industrial use as solvents, flavors, fragrances, and biofuels. The recombinant C. thermocellum engineered with deletion of carbohydrate esterases and stable overexpression of alcohol acetyltransferases improved ester production without compromised deacetylation activities. These esterases were discovered to exhibit promiscuous thioesterase activities and their deletion enhanced ester production by rerouting the electron and carbon metabolism. Ester production was further improved up to 80-fold and ester composition could be modulated by deleting lactate biosynthesis and using poplar with different pretreatment severity.

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Non-conventional yeasts, i.e. yeasts different from Saccharomyces cerevisiae, represent heterogenous group of unicellular fungi consisting of near 1500 species. Some of these species have interesting and sometimes unique properties like ability to grow on methanol, n-alkanes, ferment pentose sugars xylose and l-arabinose, grow at high temperatures (50°Ð¡ and more), overproduce riboflavin (vitamin B2) and others. These unique properties are important for development of basic science; moreover, some of them possess also significant applied interest for elaboration of new biotechnologies. Current paper represents review of the recent own results and of those of other authors in the field of non-conventional yeast study for construction of the advanced producers of biofuels (ethanol, isobutanol) from lignocellulosic sugars glucose and xylose or crude glycerol (Ogataea polymorpha, Magnusiomyces magnusii) and vitamin B2 (riboflavin) from glucose and cheese whey (Candida famata).

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Isobutanol is a potential biofuel, and its microbial production systems have demonstrated promising results. In a microbial system, the isobutanol produced is secreted into the media; however, the cells remaining after fermentation cannot be used efficiently during the isobutanol recovery process and are discarded as waste. To address this, we aimed to investigate the strategy of utilizing these remaining cells by combining the isobutanol production system with the indigo production system, wherein the product accumulates intracellularly. Accordingly, we constructed E. coli systems with genes, such as acetolactate synthase gene (alsS), ketol-acid reductoisomerase gene (ilvC), dihydroxyl-acid dehydratase (ilvD), and alpha-ketoisovalerate decarboxylase gene (kivD), for isobutanol production and genes, such as tryptophanase gene (tnaA) and flavin-containing monooxygenase gene (FMO), for indigo production. This system produced isobutanol and indigo simultaneously while accumulating indigo within cells. The production of isobutanol and indigo exhibited a strong linear correlation up to 72 h of production time; however, the pattern of isobutanol and indigo production varied. To our knowledge, this study is the first to simultaneously produce isobutanol and indigo and can potentially enhance the economy of biochemical production.

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The fruit-like aroma of two valine-derived volatiles, isobutanol and isobutyl acetate, has great impact on the flavour and taste of alcoholic beverages, including sake, a traditional Japanese alcoholic beverage. With the growing worldwide interest in sake, breeding of yeast strains with intracellular valine accumulation is a promising approach to meet a demand for sakes with a variety of flavour and taste by increasing the valine-derived aromas. We here isolated a valine-accumulating sake yeast mutant (K7-V7) and identified a novel amino acid substitution, Ala31Thr, on Ilv6, a regulatory subunit for acetohydroxy acid synthase. Expression of the Ala31Thr variant Ilv6 conferred valine accumulation on the laboratory yeast cells, leading to increased isobutanol production. Additionally, enzymatic analysis revealed that Ala31Thr substitution in Ilv6 decreased sensitivity to feedback inhibition by valine. This study demonstrated for the first time that an N-terminal arm conserved in the regulatory subunit of fungal acetohydroxy acid synthase is involved in the allosteric regulation by valine. Moreover, sake brewed with strain K7-V7 contained 1.5-fold higher levels of isobutanol and isobutyl acetate than sake brewed with the parental strain. Our findings will contribute to the brewing of distinctive sakes and the development of yeast strains with increased production of valine-derived compounds.

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Minicell, a small spherical form of bacterium produced by abnormal fission, possesses cytoplasmic constituents similar to those of the parental cell, except for genomic DNA. E. coli strains were engineered to produce minicells and value-added chemicals. Minicell-forming mutants showed enhanced tolerance to toxic chemicals and a higher intracellular NADH/NAD+ ratio than the wild-type. When toxic chemicals such as isobutanol, isobutyraldehyde, and isobutyl acetate were produced in this mutant, the titers increased by 67 %, 175 %, and 214 %, respectively. In addition, morphological changes and membrane dispersion mechanisms in minicell-forming mutants improved lycopene production by 259 %. This increase in production capacity was more pronounced when biomass hydrolysate was used as the substrate. Isobutanol and lycopene production also increased by 92 % and 295 %, respectively, on using the substrate in the mutant. It suggests that minicell-forming mutants are an excellent platform for biochemical production.

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Enzyme-catalyzed reaction cascades play an increasingly important role for the sustainable manufacture of diverse chemicals from renewable feedstocks. For instance, dehydratases from the ilvD/EDD superfamily have been embedded into a cascade to convert glucose via pyruvate to isobutanol, a platform chemical for the production of aviation fuels and other valuable materials. These dehydratases depend on the presence of both a Fe-S cluster and a divalent metal ion for their function. However, they also represent the rate-limiting step in the cascade. Here, catalytic parameters and the crystal structure of the dehydratase from Paralcaligenes ureilyticus (PuDHT, both in presence of Mg2+ and Mn2+ ) were investigated. Rate measurements demonstrate that the presence of stoichiometric concentrations Mn2+ promotes higher activity than Mg2+ , but at high concentrations the former inhibits the activity of PuDHT. Molecular dynamics simulations identify the position of a second binding site for the divalent metal ion. Only binding of Mn2+ (not Mg2+ ) to this site affects the ligand environment of the catalytically essential divalent metal binding site, thus providing insight into an inhibitory mechanism of Mn2+ at higher concentrations. Furthermore, in silico docking identified residues that play a role in determining substrate binding and selectivity. The combined data inform engineering approaches to design an optimal dehydratase for the cascade.

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Saccharomyces cerevisiae is the dominant fermentative producer of ethanol in industry and a preferred host for production of other biofuels. That said, rewiring the metabolism of S. cerevisiae to produce other fermentation products, such as isobutanol, remains an academic challenge. Many studies report aerobic production of isobutanol, but ethanol remains a substantial by-product under these conditions due to the Crabtree effect. These studies indicate that the native isobutanol pathway is incapable of carrying sufficient flux to displace ethanol. In this report, we screened a combinatorial library of pathway enzymes to identify an isobutanol pathway cassette capable of supporting the growth of a non-ethanol producing S. cerevisiae. We began by identifying a diverse set of isobutanol pathway enzyme homologs and combined each open reading frame with varied-strength promoters in a combinatorial, pooled fashion. We applied a growth-coupled screen where a functional isobutanol pathway restored NAD+ regeneration during glucose catabolism that is otherwise repressed via the Crabtree effect. Using this screen, we isolated a cassette consisting of a mosaic of bacterial and cytosol-localized fungal enzymes that conferred under aerobic conditions the ability to produce 364 mg/L isobutanol (8.8% of the theoretical maximum yield). We next shifted the cofactor usage of the isolated ketol-acid reductoisomerase enzyme in the cassette from NADPH to NADH-preferring to improve redox balance. The approach used herein isolated isobutanol producing strains that approach the best in the literature without producing substantial ethanol titers. Still, the best isolated cassette was insufficient to support anaerobic growth in the absence of ethanol fermentation - indicating the presence of further fundamental gaps in our understanding of yeast fermentation.

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The production of the biofuel, isobutanol, in E. coli faces limitations due to alcohol toxicity, product inhibition, product recovery, and long-term industrial feasibility. Here we demonstrate an approach of combining both in vivo with in vitro metabolic engineering to produce isobutanol. The in vivo production of α-ketoisovalerate (KIV) was conducted through CRISPR mediated integration of the KIV pathway in bicistronic design (BCD) in E. coli and inhibition of competitive valine pathway using CRISPRi technology. The subsequent in vitro conversion to isobutanol was carried out with engineered enzymes for 2-ketoacid decarboxylase (KIVD) and alcohol dehydrogenase (ADH). For the in vivo production of KIV and subsequent in vitro production of isobutanol, this two-step serial approach resulted in yields of 56% and 93%, productivities of 0.62 and 0.074 g L-1 h-1, and titers of 5.6 and 1.78 g L-1, respectively. Thus, this combined biosynthetic system can be used as a modular approach for producing important metabolites, like isobutanol, without the limitations associated with in vivo production using a consolidated bioprocess.

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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 ($317 MM) and OPEX ($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 $1.38/kg isobutanol. Furthermore, a single sensitivity analysis with nine different parameters was carried out for the production of CO2-derived isobutanol. The annual plant capacity, gas utilization rate, and substrate cost are the three most important economic-driving forces on the MISP of CO2-derived isobutanol. Finally, a multiple-point sensitivity analysis considering all five parameters simultaneously was performed using ideal targets, which presented the lowest 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.