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Production of 1-butanol and ethanol by recombinant E. coli strains JCL299 expressing CoA-dependent 1-butanol pathway with YqhD from E. coli and Bldh from different organisms. Dashed line represents the baseline production by using AdhE2. Detailed production procedure is listed in SI Text.
Source publication
While conservation of ATP is often a desirable trait for microbial production of chemicals, we demonstrate that additional consumption of ATP may be beneficial to drive product formation in a nonnatural pathway. Although production of 1-butanol by the fermentative coenzyme A (CoA)-dependent pathway using the reversal of β-oxidation exists in nature...
Context in source publication
Context 1
... Bldh, we searched by homology and cloned additional Bldh-like enzymes from various organisms including C. saccharoperbutylacetonicum NI-4, C. saccharobutylicum ATCC BAA-117, Geobacillus thermoglucosidasius, Clostridium kluyveri, and E. coli. We assessed the performance of these Bldh's by 1-butanol production in recombinant E. coli. As shown in Fig. 5, the E. coli strain expressing C. saccharoperbutylacetonicum NI-4 Bldh along with rest of the CoA 1-butanol pathway produced the highest titer of 1-butanol, exceeding the 1-butanol produced by E. coli strain expressing AdhE2 by nearly ...
Citations
... Clostridium strains are strict anaerobic bacteria. In the butanol production pathway, the hydrogenation of crotonyl-CoA to butyryl-CoA, catalyzed by the butyryl-CoA dehydrogenase/electron transferring flavoprotein (bcd/etfAB) complex, is difficult to express in recombinant systems (presumably due to oxygen sensitive) [39]. In addition, oxidative conditions negatively affect the reducing power, which is crucial for alcohol production. ...
... In such cases, ATP consumption can be artificially boosted to trigger favorable metabolic responses while limiting proliferation [32]. Artificially increasing ATP consumption is a promising metabolic engineering strategy for enhancing microbial metabolite production [20,26,27,[33][34][35][36][37][38][39]. For instance, engineered ATP consumption was incorporated into n-butanediol production in cyanobacteria to boost metabolic flux to target pathways [35]. ...
... Artificially increasing ATP consumption is a promising metabolic engineering strategy for enhancing microbial metabolite production [20,26,27,[33][34][35][36][37][38][39]. For instance, engineered ATP consumption was incorporated into n-butanediol production in cyanobacteria to boost metabolic flux to target pathways [35]. More recently, introducing E. coli F1-ATPase expression in S. cerevisiae led to improved ethanol production [18]. ...
Background
“ATP wasting” has been observed in ¹³C metabolic flux analyses of Saccharomyces cerevisiae, a yeast strain commonly used to produce ethanol. Some strains of S. cerevisiae, such as the sake strain Kyokai 7, consume approximately two-fold as much ATP as laboratory strains. Increased ATP consumption may be linked to the production of ethanol, which helps regenerate ATP.
Results
This study was conducted to enhance ethanol and 2,3-butanediol (2,3-BDO) production in the S. cerevisiae strains, ethanol-producing strain BY318 and 2,3-BDO-producing strain YHI030, by expressing the fructose-1,6-bisphosphatase (FBPase) and ATP synthase (ATPase) genes to induce ATP dissipation. The introduction of a futile cycle for ATP consumption in the pathway was achieved by expressing various FBPase and ATPase genes from Escherichia coli and S. cerevisiae in the yeast strains. The production of ethanol and 2,3-BDO was evaluated using high-performance liquid chromatography and gas chromatography, and fermentation tests were performed on synthetic media under aerobic conditions in batch culture. The results showed that in the BY318-opt_ecoFBPase (expressing opt_ecoFBPase) and BY318-ATPase (expressing ATPase) strains, specific glucose consumption was increased by 30% and 42%, respectively, and the ethanol production rate was increased by 24% and 45%, respectively. In contrast, the YHI030-opt_ecoFBPase (expressing opt_ecoFBPase) and YHI030-ATPase (expressing ATPase) strains showed increased 2,3-BDO yields of 26% and 18%, respectively, and the specific production rate of 2,3-BDO was increased by 36%. Metabolomic analysis confirmed the introduction of the futile cycle.
Conclusion
ATP wasting may be an effective strategy for improving the fermentative biosynthetic capacity of S. cerevisiae, and increased ATP consumption may be a useful tool in some alcohol-producing strains.
... The combination of ATP hydrolysis, bicarbonate fixation and direct decarboxylation together make this route thermodynamically far superior to the linear PhaA dependent route. The use of NphT7 in combination with Acc already demonstrated to support production of isobutanol (Lan and Liao, 2012) and 3-hydroxybutyrate (Ku and Lan, 2018) in the cyanobacterium Synechococcus elongatus. Whereas in C. necator the production of NphT7 requires heterologous gene expression, the Acc complex is endogenous and represents the first step of fatty acid biosynthesis. ...
... This architecture fits well with the requirement of a formatotrophic growth mode, where oxidation of formate via Fdh increases the NADH pool, with consequent lower demand of flux through the TCA cycle via acetyl-CoA. Therefore, the use of this bypass (at the cost of one ATP investment) confirmed to be a valuable strategy for circumventing thermodynamic bottlenecks when the intracellular pool of acetyl-CoA is limited (Lan and Liao, 2012;Orsi et al., 2022). Under formatotrophic growth conditions, the malonyl-CoA bypass did not show growth deficit compared to the control strain with the direct route from acetyl-CoA to acetoacetyl-CoA. ...
To advance the sustainability of the biobased economy, our society needs to develop novel bioprocesses based on truly renewable resources. The C1-molecule formate is increasingly proposed as carbon and energy source for microbial fermentations, as it can be efficiently generated electrochemically from CO2 and renewable energy. Yet, its biotechnological conversion into value-added compounds has been limited to a handful of examples. In this work, we engineered the natural formatotrophic bacterium C. necator as cell factory to enable biological conversion of formate into crotonate, a platform short-chain unsaturated carboxylic acid of biotechnological relevance. First, we developed a small-scale (150-mL working volume) cultivation setup for growing C. necator in minimal medium using formate as only carbon and energy source. By using a fed-batch strategy with automatic feeding of formic acid, we could increase final biomass concentrations 15-fold compared to batch cultivations in flasks. Then, we engineered a heterologous crotonate pathway in the bacterium via a modular approach, where each pathway section was assessed using multiple candidates. The best performing modules included a malonyl-CoA bypass for increasing the thermodynamic drive towards the intermediate acetoacetyl-CoA and subsequent conversion to crotonyl-CoA through partial reverse β-oxidation. This pathway architecture was then tested for formate-based biosynthesis in our fed-batch setup, resulting in a two-fold higher titer, three-fold higher productivity, and five-fold higher yield compared to the strain not harboring the bypass. Eventually, we reached a maximum product titer of 148.0 ± 6.8 mg/L. Altogether, this work consists in a proof-of-principle integrating bioprocess and metabolic engineering approaches for the biological upgrading of formate into a value-added platform chemical.
... Taken together, PhaA is a key determinant of the bioplastic production with these bacteria. Essentially, the condensation reaction of acetyl-CoA is thermodynamically unfavorable and is not oriented toward the formation of acetoacetyl-CoA [39]. Therefore, it makes sense to use CoaA to fill the intracellular acetyl-CoA pool and apply pressure in the direction of acetoacetyl-CoA production. ...
Background
Coenzyme A (CoA) is a carrier of acyl groups. This cofactor is synthesized from pantothenic acid in five steps. The phosphorylation of pantothenate is catalyzed by pantothenate kinase (CoaA), which is a key step in the CoA biosynthetic pathway. To determine whether the enhancement of the CoA biosynthetic pathway is effective for producing useful substances, the effect of elevated acetyl-CoA levels resulting from the introduction of the exogenous coaA gene on poly(3-hydroxybutyrate) [P(3HB)] synthesis was determined in Escherichia coli, which express the genes necessary for cyanobacterial polyhydroxyalkanoate synthesis (phaABEC).
Results
E. coli containing the coaA gene in addition to the pha genes accumulated more P(3HB) compared with the transformant containing the pha genes alone. P(3HB) production was enhanced by precursor addition, with P(3HB) content increasing from 18.4% (w/w) to 29.0% in the presence of 0.5 mM pantothenate and 16.3%–28.2% by adding 0.5 mM β-alanine. Strains expressing the exogenous coaA in the presence of precursors contained acetyl-CoA in excess of 1 nmol/mg of dry cell wt, which promoted the reaction toward P(3HB) formation. The amount of acetate exported into the medium was three times lower in the cells carrying exogenous coaA and pha genes than in the cells carrying pha genes alone. This was attributed to significantly enlarging the intracellular pool size of CoA, which is the recipient of acetic acid and is advantageous for microbial production of value-added materials.
Conclusions
Enhancing the CoA biosynthetic pathway with exogenous CoaA was effective at increasing P(3HB) production. Supplementing the medium with pantothenate facilitated the accumulation of P(3HB). β-Alanine was able to replace the efficacy of adding pantothenate.
... The predominant volatiles emitted after HS were alcohol and acid derivatives of butane: 3-methoxy-2-butanol (Fig. 5C), S-t-butyl ester of 5-oxohexanethioic acid (Fig. 5D), 3-methyland 2-methylbutanoic acids (Table S5). Their production has been described in bacteria after anaerobic glycolysis and their occurrence in plants is unknown (Lan and Liao, 2012). Nevertheless, 3-methylbutanoic acid, known as isovaleric acid, has been found to inhibit plant growth (Murata et al., 2022). ...
Heat stress is a frequent environmental constraint. Phytohormones can significantly affect plant thermotolerance. This study compares the effects of exogenous cytokinin meta-topolin-9-(tetrahydropyran-2-yl)purine (mT9THP) on rice (Oryza sativa) under control conditions, after acclimation by moderate temperature (A; 37 °C, 2h), heat stress (HS; 45 °C, 6h) and their combination (AHS). mT9THP is a stable cytokinin derivative that releases active meta-topolin gradually, preventing the rapid deactivation reported after exogenous cytokinin application. Under control conditions, mT9THP negatively affected jasmonic acid in leaves and abscisic and salicylic acids in crowns (meristematic tissue crucial for tillering). Exogenous cytokinin stimulated the emission of volatile organic compounds (VOC), especially 2,3-butanediol. Acclimation upregulated trans-zeatin, expression of stress- and hormone-related genes, and VOC emission. The combination of acclimation and mT9THP promoted the expression of stress markers and antioxidant enzymes and moderately increased VOC emission, including 2-ethylhexyl salicylate or furanones. AHS and HS responses shared some common features, namely, increase of ethylene precursor aminocyclopropane-1-carboxylic acid (ACC), cis-zeatin and cytokinin methylthio derivatives, as well as the expression of heat shock proteins, alternative oxidases, and superoxide dismutases. AHS specifically induced jasmonic acid and auxin indole-3-acetic acid levels, diacylglycerolipids with fewer double bonds, and VOC emissions [e.g., acetamide, lipoxygenase (LOX)-derived volatiles]. Under direct HS, exogenous cytokinin mimicked some positive acclimation effects. The combination of mT9THP and AHS had the strongest thermo-protective effect, including a strong stimulation of VOC emissions (including LOX-derived ones). These results demonstrate for the first time the crucial contribution of volatiles to the beneficial effects of cytokinin and AHS on rice thermotolerance.
... Researchers successfully used CO 2 as a substrate to produce butanol from cyanobacteria. Photosynthetic butanol production was achieved by introducing a modified CoA-dependent butanol production pathway to the cyanobacteria Synechococcus elongatus PCC7942 [103,104]. Likewise, exogenous synthetic genes for 2,3-BD were introduced into cyanobacteria with appropriately controlled promoters and operons to direct the synthesis of 2,3-BD from CO 2 [105]. ...
Pyruvate is a hub of various endogenous metabolic pathways, including glycolysis, TCA cycle, amino acid, and fatty acid biosynthesis. It has also been used as a precursor for pyruvate-derived compounds such as acetoin, 2,3-butanediol (2,3-BD), butanol, butyrate, and L-alanine biosynthesis. Pyruvate and derivatives are widely utilized in food, pharmaceuticals, pesticides, feed additives, and bioenergy industries. However, compounds such as pyruvate, acetoin, and butanol are often chemically synthesized from fossil feedstocks, resulting in declining fossil fuels and increasing environmental pollution. Metabolic engineering is a powerful tool for producing eco-friendly chemicals from renewable biomass resources through microbial fermentation. Here, we review and systematically summarize recent advances in the biosynthesis pathways, regulatory mechanisms, and metabolic engineering strategies for pyruvate and derivatives. Furthermore, the establishment of sustainable industrial synthesis platforms based on alternative substrates and new tools to produce these compounds is elaborated. Finally, we discuss the potential difficulties in the current metabolic engineering of pyruvate and derivatives and promising strategies for constructing efficient producers.
... While much of the current research is focused on increasing growth rates, improving photosynthesis and carbon capture, and conferring stress tolerance, cells that accumulate high levels of ATP and glycogen in their cells, may also be promising hosts for material production. In a previous study, Lan and Liao introduced ATP-driven synthesis of acetoacetyl-CoA into S. elongatus PCC 7942 and successfully produced 1-butanol by photosynthesis 43 . Meanwhile, Hasunuma et al. reported that overexpression of flv3 enhances both ATP supply and glycogen biosynthesis, which is a promising approach to biofuel production using cyanobacteria 44 . ...
The lipid composition of thylakoid membranes is conserved from cyanobacteria to green plants. However, the biosynthetic pathways of galactolipids, the major components of thylakoid membranes, are known to differ substantially between cyanobacteria and green plants. We previously reported on a transformant of the unicellular rod-shaped cyanobacterium Synechococcus elongatus PCC 7942, namely SeGPT, in which the synthesis pathways of the galactolipids monogalactosyldiacylglycerol and digalactosyldiacylglycerol are completely replaced by those of green plants. SeGPT exhibited increased galactolipid content and could grow photoautotrophically, but its growth rate was slower than that of wild-type S. elongatus PCC 7942. In the present study, we investigated pleiotropic effects that occur in SeGPT and determined how its increased lipid content affects cell proliferation. Microscopic observations revealed that cell division and thylakoid membrane development are impaired in SeGPT. Furthermore, physiological analyses indicated that the bioenergetic state of SeGPT is altered toward energy storage, as indicated by increased levels of intracellular ATP and glycogen. We hereby report that we have identified a new promising candidate as a platform for material production by modifying the lipid synthesis system in this way.
... Butanol production was achieved when C. tyrobutyricum ATCC 25,755 was engineered to overexpress aldehyde/alcohol dehydrogenase 2 (adhE2) from C. acetobutylicum ATCC 824, which converts butyryl-CoA to butanol, under the control of native thiolase (thl) promoter . Substitution of bifunctional aldehyde/ alcohol dehydrogenase (AdhE2) with separate butyraldehyde dehydrogenase (Bldh) and NADPH-dependent alcohol dehydrogenase (YqhD) increased butanol production by fourfold in Synechococcus elongatus PCC 7942 (Lan and Liao, 2012). Modification of CoAdependent and keto acid pathway was achieved by expressing the genes ter-bdhB-bdhA and kivd, respectively in Klebsiella pneumonia. ...
The tremendous consumption of fossil fuels has demanded an ultimatum to satisfy the future necessities of energy. To fulfill, biofuels such as bioethanol and biobutanol have been gaining attention as next-generation biofuels. Besides its advantages, biobutanol demands expensive recovery/downstream processing. Recent advancements in the rapidly emerging nanotechnology field are a potential approach to producing conventional and economically sound biobutanol. It enables us to build a stable and robust Nano-biocatalyst system to encompass the production cost. Nanoparticles (NPs) being used in Nano-biocatalyst are highly promising to initiate the catalysis and to modify the chemical processes, by that means transforming both the nature and distribution of products. The use of NPs during downstream processing such as adsorption, gas stripping, liquid-liquid extraction, pervaporation, etc. are also resolving economic issues related to separation and purification techniques. Also, nanomaterials are used in Nanocomposite membranes for superior separation performance, for immobilization of expensive enzymes that allow them to be reused several times over, in biofuels to ameliorate thermos-physical properties, and in fermentation to enhance the production rate thus significantly reducing the overall processing cost. The present chapter represents an overview of biobutanol, its challenges associated, and chiefly stresses the application of nanotechnology in biobutanol production. The plethora of emerging techniques and applications of nanotechnology in different stages of biobutanol production provides a sustainable approach by lowering the biomass processing and product cost and can assist us to set up a robust system with long-term stability and reduced environmental hazards.
... Butanol production was achieved when C. tyrobutyricum ATCC 25,755 was engineered to overexpress aldehyde/alcohol dehydrogenase 2 (adhE2) from C. acetobutylicum ATCC 824, which converts butyryl-CoA to butanol, under the control of native thiolase (thl) promoter . Substitution of bifunctional aldehyde/ alcohol dehydrogenase (AdhE2) with separate butyraldehyde dehydrogenase (Bldh) and NADPH-dependent alcohol dehydrogenase (YqhD) increased butanol production by fourfold in Synechococcus elongatus PCC 7942 (Lan and Liao, 2012). Modification of CoAdependent and keto acid pathway was achieved by expressing the genes ter-bdhB-bdhA and kivd, respectively in Klebsiella pneumonia. ...
In recent decades, research has oriented toward finding a renewable fuel from biological sources due to the depletion of fossil fuel reserves and increasing awareness on global warming. Biobutanol has been recognized as a promising alternate fuel owing to its excellent combustion value, less volatility, high octane rating, and less corrosiveness. Microbial fermentation of acetone–butanol–ethanol (ABE) has been noticed as the leading industrial bioprocess for biobutanol production from biomass. Recently, algae seem to be ideal candidates for producing biobutanol because they contain different fermentable sugars, are simple in production and are cost effective as they have no lignin unlike lignocellulosic biomass. Nonetheless, commercialization of algal biobutanol to replace gasoline is still not viable due to uncertainty of selecting suitable algal species, pretreatment of biomass and conversion of sugars to butanol. Hence, research is to be focused to standardize and analyze the potentiality of algal biobutanol. This chapter elaborates on the necessity of biobutanol, existing techniques, fermentation feasibilities, and other technical obstacles to commercialize biobutanol. In addition, advances of bioprocess techniques and optimization for improved biobutanol production and recovery were also discussed.
... The microbial synthesis of higher alcohols (C 3+ fatty alcohols) is similar to the synthesis of ethanol, which is also achieved by introducing or optimizing the metabolic pathway of higher alcohols in the host. Lan and Liao (2012) promoted the reversal of β-oxidation by modifying the ATP consumption to acquire 1-butanol under the photosynthetic conditions using cyanobacteria PCC 7942. Li et al. (2012) modified Ralstonia eutropha H16 by genetic engineering technology to produce isobutanol and isopentanol. ...
Environmental problems such as greenhouse effect, the consumption of fossil energy, and the increase of human demand for energy are becoming more and more serious, which force researcher to turn their attention to the reduction of CO 2 and the development of renewable energy. Unsafety, easy to lead to secondary environmental pollution, cost inefficiency, and other problems limit the development of conventional CO 2 capture technology. In recent years, many microorganisms have attracted much attention to capture CO 2 and synthesize valuable products directly. Fatty acid derivatives (e.g., fatty acid esters, fatty alcohols, and aliphatic hydrocarbons), which can be used as a kind of environmentally friendly and renewable biofuels, are sustainable substitutes for fossil energy. In this review, conventional CO 2 capture techniques pathways, microbial CO 2 concentration mechanisms and fixation pathways were introduced. Then, the metabolic pathway and progress of direct production of fatty acid derivatives from CO 2 in microbial cell factories were discussed. The synthetic biology means used to design engineering microorganisms and optimize their metabolic pathways were depicted, with final discussion on the potential of optoelectronic–microbial integrated capture and production systems.
