Butanol Synthesis Research Articles

Comparative proteomics analysis of high n-butanol producing metabolically engineered Clostridium tyrobutyricum

The acidogenic Clostridium tyrobutyricum has recently been metabolically engineered to produce n-butanol. The objective of this study was to obtain a comprehensive understanding as to how butanol production was regulated in C. tyrobutyricum to guide the engineering of next-generation strains. We performed a comparative proteomics analysis, covering 78.1% of open reading frames and 95% of core enzymes, using wild type, ACKKO mutant (Δack) producing 37.30g/L of butyrate and ACKKO-adhE2 mutant (Δack-adhE2) producing 16.68g/L of butanol. In ACKKO-adhE2, the expression of most glycolytic enzymes was decreased, the thiolase (thl), acetyl-CoA acetyltransferase (ato), 3-hydroxybutyryl-CoA dehydrogenase (hbd) and crotonase (crt) that convert acetyl-CoA to butyryl-CoA were increased, and the heterologous bifunctional acetaldehyde/alcohol dehydrogenase (adhE2) catalyzing butanol formation was highly expressed. The apparent imbalance of energy and redox was observed due to the downregulation of acids production and the addition of butanol synthesis pathway, which also resulted in increased expression of chaperone proteins and glycerol-3-phosphate dehydrogenase (glpA) and the silence of sporulation transcription factor Spo0A (spo0A) as the cellular responses to butanol production. This study revealed the mechanism of carbon redistribution, and limiting factors and rational metabolic cell and process engineering strategies to achieve high butanol production in C. tyrobutyricum.

Metabolic flux and transcriptional analysis elucidate higher butanol/acetone ratio feature in ABE extractive fermentation by Clostridium acetobutylicum using cassava substrate

In acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum ATCC 824 using corn-based substrate, the solvents are generally produced at a ratio of 3:6:1 (A:B:E, w/w). A higher butanol/acetone ratio of 2.9:1 was found when cassava was used as the substrate of an in-situ extractive fermentation by C. acetobutylicum. This ratio had a 64% increment compared to that on corn-based substrate. The metabolic flux and (key enzymes) genes transcriptional analysis indicated that weakened metabolic fluxes in organic acids (especially butyrate) formation and re-assimilation pathways, which associated with lower buk and ctfAB transcriptional levels, contributed to higher butanol and lower acetone production rate in fermentations on cassava. Moreover, NADH generation was enhanced under the enriched reductive environment of using cassava-based substrate, which converted more carbon flux to butanol synthesis pathway, also leading to a higher ratio of butanol/acetone. To further increase butanol/acetone ratio, tiny amount of electron carrier, neutral red was supplemented into cassava-based substrate at 60 h when butonal production rate reached maximal level. However, neutral red addition enhanced NADH production, followed with strengthening the metabolic fluxes of organic acids formation/re-assimilation pathways, resulted in unchanged in butanol/acetone ratio. A further increase in butanol/acetone ratio could be realized when NADH regeneration was enhanced and the metabolic fluxes in organic acids formation/reutilization routes were controlled at suitably low levels simultaneously.

A synthetic O2 -tolerant butanol pathway exploiting native fatty acid biosynthesis in Escherichia coli.

Several synthetic metabolic pathways for butanol synthesis have been reported in Escherichia coli by modification of the native CoA-dependent pathway from selected Clostridium species. These pathways are all dependent on the O2 -sensitive AdhE2 enzyme from Clostridium acetobutylicum that catalyzes the sequential reduction of both butyryl-CoA and butyraldehyde. We constructed an O2 -tolerant butanol pathway based on the activities of an ACP-thioesterase, acting on butyryl-ACP in the native fatty acid biosynthesis pathway, and a promiscuous carboxylic acid reductase. The pathway was genetically optimized by screening a series of bacterial acyl-ACP thioesterases and also by modification of the physical growth parameters. In order to evaluate the potential of the pathway for butanol production, the ACP-dependent butanol pathway was compared with a previously established CoA-dependent pathway. The effect of (1) O2 -availability, (2) media, and (3) co-expression of aldehyde reductases was evaluated systematically demonstrating varying and contrasting functionality between the ACP- and CoA-dependent pathways. The yield of butanol from the ACP-dependent pathway was stimulated by enhanced O2 -availability, in contrast to the CoA-dependent pathway, which did not function well under aerobic conditions. Similarly, whilst the CoA-dependent pathway only performed well in complex media, the ACP-dependent pathway was not influenced by the choice of media except in the absence of O2 . A combination of a thioesterase from Bacteroides fragilis and the aldehyde reductase, ahr, from E. coli resulted in the greatest yield of butanol. A product titer of ~300 mg/L was obtained in 24 h under optimal batch growth conditions, in most cases exceeding the performance of the reference CoA-pathway when evaluated under equivalent conditions.

Meiothermus ruber thiolase – A new process stable enzyme for improved butanol synthesis

Butanol is an important renewable building block for the chemical, textile, polymer and biofuels industry due to its increased energy density. Current biotechnological butanol production is a Clostridial based anaerobic fermentation process. Thiolase (EC 2.3.1.9/EC 2.3.1.16) is a key enzyme in this biosynthetic conversion of glucose to butanol. It catalyzes the condensation of two acetyl-CoA molecules, forming acetoacetyl-CoA, which is the first committed step in butanol biosynthesis. The well characterized clostridial thiolases are neither solvent nor thermo stable, which limits butanol yields. We have isolated and characterized a new thermo- (IT50 50 °C = 199 ± 0.1 h) and solvent stable (IS50 > 4%) thiolase derived from the thermophilic bacterium Meiothermus ruber. The observed catalytic constants were Km = 0.07 ± 0.01 mM and kcat = 0.80 ± 0.01 s(-1). In analogy to other thiolases, the enzyme was inhibited by NAD(+) (Ki = 38.7 ± 5.8 mM) and CoA (Ki = 105.1 ± 6.6 μM) but not NADH. The enzyme was stable under harsh process conditions (T = 50 °C, Butanol = 4% v/v) for prolonged time periods (τ = 7 h). The new enzyme provides for targeted in-vivo and in-vitro butanol biosynthesis under industrially relevant process conditions.

Biobutanol as an alternative type of fuel

Butanol is an alternative type of fuel. Amid the depletion of the world’s (accessible) petroleum reserves, butanol is considered to be a potential energy source. Although butanol production was initially associated with microbiological synthesis, on an industrial scale, butanol is produced via chemical synthesis. For its production to become economically viable, strains of microorganisms must have a potential for excessive synthesis of butanol. The pathways of butanol synthesis via microorganisms and their regulation, as well as the most promising producer strains for industrial production and methods for increasing their productivity, are discussed in the survey.

Isolation and screening of carboxydotrophs isolated from composts and their potential for butanol synthesis

Carboxydotrophs are known for their ability to convert carbon monoxide (CO) to butanol through fermentation. Such a platform offers a promising alternative approach to biofuel production from synthesis gas feedstocks. In this study, carboxydotrophs were isolated from various manure compost. Out of 500 isolates, only 11 carboxydotrophs (7 mesophiles and 4 thermophiles) were found to utilize CO as the sole source of carbon and energy. To assess the biochemical basis for their ability to produce biofuel (butanol), the level of activities of CO dehydrogenase (CODH), hydrogenase and butanol dehydrogenase (BDH) enzymes for these isolates against the known carboxydotroph, Butyribacterium methylotrophicum was assessed. All isolates showed evidence of enzyme activities (0.16–2.20 μmol min−1), with the majority exhibiting higher activities compared with the known carboxydotroph, B. methylotrophicum (0.33–0.71 μmol min−1). The level of activities for CODH and BDH ranged from 0.163–3.59 μmol min−1 and 0.19–2.2 μmol min−1, respectively. Three isolates (M7-1, T2-22, and T3-14) demonstrated enzymatic activity three to seven times higher than B. methylotrophicum. Of these, T2-22 exhibited the highest BDH activity and shows great promise in the conversion of toxic CO into butanol more so than other carboxytotrophs known thus far. This study revealed some biochemical basis for butanol production from CO by carboxydotrophs. However, more research is needed to discover a direct biological route for butanol production from CO to strengthen their potential for synthesis gas bioprocessing. Follow-up work will focus on whole-genome sequencing of the promising isolate T2-22 to provide system-level insights into how carboxydotrophs utilize and regulate their molecular machineries for butanol production.

Evaluation of Carbon and Electron Flow in <i>Lactobacillus brevis</i> as a Potential Host for Heterologous 1-Butanol Biosynthesis

Heterofermentative lactic acid bacterium Lactobacillus brevis may be considered as a promising host for heterologous butanol synthesis because of tolerance to butanol and ability to ferment pentose and hexose sugars from wood hydrolysates that are cheap and renewable carbohydrate source. Carbon and electron flow was evaluated in two L. brevis strains in order to assess metabolic potential of these bacteria for heterologous butanol synthesis. Conditions required for generation of acetyl-CoA and NADH which are necessary for butanol biosynthesis have been determined. Key enzymes controlling direction of metabolic fluxes in L. brevis in various redox conditions were defined. In anaerobic glucose fermentation, the carbon flow through acetyl-CoA is regulated by aldehyde dehydrogenase ALDH possessing low affinity to NADH and activity (KmNADH= 200 μM, Vmax= 0.03 U/mg of total cell protein). Aerobically, the NADH-oxidase NOX (KmNADH= 25 μM, Vmax = 1.7 U/mg) efficiently competes with ALDH for NADH that results in formation of acetate instead of acetyl-CoA. In general, external electron acceptors (oxygen, fructose) and pentoses decrease NADH availability for native ethanol and recombinant butanol enzymes and therefore reduce carbon flux through acetyl-CoA. Pyruvate metabolism was studied in order to reveal redirection possibilities of competitive carbon fluxes towards butanol synthesis. The study provides a basis for the rational development of L. brevis strains producing butanol from wood hydrolysate.

Yeast extract promotes phase shift of bio-butanol fermentation by Clostridium acetobutylicum ATCC824 using cassava as substrate

When fermenting on cassava (15–25%, w/v) with Clostridium acetobutylicum ATCC824, a severe delay (18–40h) was observed in the phase shift from acidogenesis to solventogenesis, compared to the cases of fermenting on corn. By adding yeast extract (2.5g/L-broth) into cassava meal medium when the delay appeared, the phase shift was triggered and fermentation performances were consequently improved. Total butanol concentrations/butanol productivities, compared to those with cassava substrate alone, increased 15%/80% in traditional fermentation while 86%/79% in extractive fermentation using oleyl alcohol as the extractant, and reached the equivalent levels of those using corn substrate. Analysis of genetic transcriptional levels and measurements of free amino acids in the broth demonstrated that timely and adequate addition of yeast extract could promote phase shift by increasing transcriptional level of ctfAB to 16-fold, and indirectly enhance butanol synthesis through accelerating the accumulation of histidine and aspartic acid families.

RETOOLING A BACTERIAL BIOFUEL FACTORY

IN A STEP TOWARD more efficient biofuel production, two independent teams have improved on past efforts to engineer Escherichia coli so the microbe produces higher outputs of n -butanol than ever before—more than 30 times as much as prior engineered strains. The studies were presented at last month’s American Chemical Society national meeting in Anaheim, Calif. Several chemical companies are pursuing butanol as a biobased fuel source. Most vehicles can run on a blend of butanol and petroleum-based gasoline. Rising gas prices and modern biotechnology have revived interest in an early-20th-century butanol fermentation process. That method used a slow-growing bacterium called Clostridium acetobutylicum and was best known for producing butanol as fuel for World War II-era planes. Researchers previously transferred Clostridium’ s genes for butanol synthesis into the workhorse bacterium E. coli, but butanol output has been modest. In the Division of Organic Chemistry, graduate student Brooks B. Bond-Watts prese...

Selective Butanol Synthesis over Rhodium−Molybdenum Catalysts Supported in Ordered Mesoporous Silica

Rhodium catalysts were synthesized in ordered mesoporous silica FSM-16 using Rh/Al heteropolyacid anions and/or [Rh(COD) 2 ] + complex. The hydroformylation activity of propene was compared to impregnated Rh/ FSM-16 catalysts prepared from rhodium chloride. The catalysts were very selective to produce butanols due to the effect of two-dimensional mesoporous reaction space (effective internal pore diameter 27 A). The selectivity to butanols (n-butanol and i-butanol) was as much as >98% over [RhMo 6 O 18 (OH) 6 ] 3- /FSM-16 catalysts and 73% over RhCl 3 /FSM-16 catalysts. Under the hydroformylation reaction conditions at 433 K and 60 kPa, 22-28 A of supported nanoparticles were present based on Rh K-edge extended X-ray absorption fine structure (EXAFS) and high-resolution transmission electron microscopy (HR TEM) measurements. Rh metallic nanoparticles atomically mixed with Mo atoms and distorted heteropolyacid species coexisted in the [RhMo 6 O 18 (OH) 6 ] 3- /FSM-16 catalysts based on Rh and Mo K-edge EXAFS and HR TEM measurements. The population of metallic nanoparticles increased as the metal loading amount decreased from 5.2 to 0.22 wt % Rh. Thus, metallic nanoparticles in FSM-16 were essential for the butanol synthesis and Mo played an additional promoter role to increase the selectivity further. A reaction mechanism was proposed in which the metallic nanoparticle surface was predominantly adsorbed with CO and multiple-step hydrogenations of oxygenate intermediates proceeded to form butanols during slow diffusion in the two-dimensional mesoporous space.

Initiation of endospore formation in Clostridium acetobutylicum

Endospore formation in bacilli and clostridia shows remarkable similarities in morphology as well as in physiological and molecular biological cellular events. Major differences are the formation of clostridial stage cells and granulose accumulation in clostridia. In both genera, a cascade of sigma factors is activated after septation (by help of σ H and Spo0A∼P) in the sequence σ F, σ E, σ G, and σ K. Of these, σ F and σ G are active inside the forespore and are regulated by anti-sigma factors and anti-anti-sigma factors, whereas σ E and σ K (mother cell-specific sigma factors) are synthesized as precursor proteins and activated by proteolysis. Each of these sigma factors allows transcription of a specific set of genes and operons, thus leading to the orchestral expression of stage-specific proteins required for successful sporulation. Both, the genetic organization of the respective operons and the expression pattern of the sigma factors are very similar in Bacillus subtilis and Clostridium acetobutylicum, the model organisms of the two genera. However, a major regulatory difference is found in initiation of endospore formation. Genome sequencing revealed that clostridia do not contain components of the so-called phosphorelay, with the exception of the essential transcription factor Spo0A. This might reflect recognition of different environmental signals, as for clostridia nutrient limitation is no prerequisite for sporulation. In contrast to Bacillus, the clostridial sigH gene is constitutively expressed at a low level, with no increase at the onset of spore formation. The spo0A gene in C. acetobutylicum is also constitutively expressed, but Spo0A synthesis only occurs during the early and mid-exponential growth phase, indicating a posttranscriptional or cotranslational regulation. Mutational studies revealed an important regulatory function of a dual palindrome region upstream of the spo0A gene of C. acetobutylicum, part of which overlaps with a Spo0A-binding site. In addition to controlling sporulation genes, phosphorylated clostridial Spo0A is involved in regulation of acetone and butanol synthesis.

The improvement of glucose/xylose fermentation by Clostridium acetobutylicum using calcium carbonate

Batch fermentation of 60g/l glucose/xylose mixture by Clostridium acetobutylicum ATCC 824 was investigated on complex culture medium. Different proportions of mixtures, ranged between 10 and 50g of each sugar/l, were fermented during pH control at 4.8 (optimum pH for solventogenesis) or during CaCO3 addition. Using xylose-pregrown cells and pH control, an important amount of xylose was left over at the end of the fermentation when the glucose concentration was higher than that of xylose. The addition of 10g of CaCO3/l (to prevent the pH dropping below 4.8) increased xylose uptake: a substantial decrease of residual xylose was observed when xylose-pregrown cells as well as glucose-pregrown cells were used as inoculum for all the mixture proportions studied. MgCO3 (Mg2+-containing compound) and CaCl2 (Ca2+-containing compound) reduced residual xylose only during pH control at 4.8 by NaOH addition. As butanol is the major limiting factor of xylose uptake in C. acetobutylicum, fermentations were carried out with or without CaCO3 in butanol-containing media or in iron deficient media (under iron limitation, butanol synthesis occurred early and could inhibit xylose uptake). Results showed that an excess of CaCOCaCO3 could increase butanol tolerance which resulted in an increase in xylose utilization. This positive effect seem to be specific to Ca2+- or Mg2+-containing compounds, going beyond the buffering effect of carbonate.

Solventogenic enzymes of Clostridium acetobutylicum: catalytic properties, genetic organization, and transcriptional regulation.

The enzymes acetoacetate decarboxylase and coenzyme A transferase catalyse acetone production from acetoacetyl-CoA in Clostridium acetobutylicum. The adc gene encoding the former enzyme is organized in a monocistronic operon, while the ctf genes form a common transcription unit with the gene (adhE) encoding a probable polyfunctional aldehyde/alcohol dehydrogenase. This genetic arrangement could reflect physiological requirements at the onset of solventogenesis. In addition to AdhE, two butanol dehydrogenase isozymes and a thiolase are involved in butanol synthesis. RNA analyses showed a sequential order of induction for the different butanol dehydrogenase genes, indicating an in vivo function of BdhI in low level butanol formation. The physiological roles of AdhE and BdhII most likely involve high level butanol formation, with AdhE being responsible for the onset of solventogenesis and BdhII ensuring continued butanol production. Addition of methyl viologen results in artificially induced butanol synthesis which seems to be mediated by a still unknown set of enzymes. Although the signal that triggers the shift to solventogenesis has not yet been elucidated, recent investigations suggest a possible function of DNA supercoiling as a transcriptional sensor of the respective environmental stimuli.

Solventogenic enzymes of Clostridium acetobutylicum: catalytic properties, genetic organization, and transcriptional regulation

The enzymes acetoacetate decarboxylase coenzyme A transferase catalyse acetone production from acetoacetyl-CoA in Clostridium acetobutylicum. The adc gene encoding the former enzyme is organized in a monocistronic operon, while the ctf genes form a common transcription unit with the gene ( adhE) encoding a probable polyfunctional aldehyde/alcohol dehydrogenase. This genetic arrangement could reflect physiological requirements at the onset of solventogenesis. In addition to AdhE, two butanol dehydrogenase isozymes and a thiolase are involved in butanol synthesis. RNA analyses showed a sequential order of induction for the different butanol dehydrogenase genes, indicating an in vivo function of BdhI in low level butanol formation. The physiological roles of AdhE and BdhII most likely involve high level butanol formation, with AdhE being responsible for the onset of solventogenesis and BdhII ensuring continued butanol production. Addition of methyl viologen results in artifically induced butanol synthesis which seems to be mediated by a still unknown set of enzymes. Although the signal that triggers the shift to solventogenesis has not yet been elucidated, recent investigations suggest a possible function of DNA supercoiling as a transcriptional sensor of the respective environmental stimuli.

Treatment with allyl alcohol selects specifically for mutants ofClostridium acetobutylicumdefective in butanol synthesis

Treatment of Clostridium acetobutylicum with allyl alcohol allowed the selection of mutants which were unable to produce n-butanol, whereas the synthesis of acetone and ethanol was unaffected. Enzymatic investigations revealed that all mutant strains were devoid of butyraldehyde dehydrogenase or showed a very low activity of this enzyme as compared to the wild type.

Treatment with allyl alcohol selects specifically for mutants of Clostridium acetobutylicum defective in butanol synthesis

Treatment with allyl alcohol selects specifically for mutants of Clostridium acetobutylicum defective in butanol synthesis