scholarly journals Metabolic Engineering of Clostridium acetobutylicum ATCC 824 for Isopropanol-Butanol-Ethanol Fermentation

2011 ◽  
Vol 78 (5) ◽  
pp. 1416-1423 ◽  
Author(s):  
Joungmin Lee ◽  
Yu-Sin Jang ◽  
Sung Jun Choi ◽  
Jung Ae Im ◽  
Hyohak Song ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Clostridium Acetobutylicum ◽  
Secondary Alcohol ◽  
Clostridium Beijerinckii ◽  
Fuel Additive ◽  
Gas Stripping ◽  
Alcohol Fermentation ◽  
Content Type ◽  
Formation Pathway ◽  
Total Alcohol

ABSTRACTClostridium acetobutylicumnaturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineeredC. acetobutylicumcan be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene fromClostridium beijerinckiiNRRL B-593 (i.e.,adhB-593) inC. acetobutylicumATCC 824. To increase the total alcohol titer, a synthetic acetone operon (actoperon;adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in thebukgene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged byin situremoval of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.

scholarly journals A Quantitative System-Scale Characterization of the Metabolism of Clostridium acetobutylicum

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Minyeong Yoo ◽  
Gwenaelle Bestel-Corre ◽  
Christian Croux ◽  
Antoine Riviere ◽  
Isabelle Meynial-Salles ◽  
...  
Keyword(s):  
Steady State ◽  
Metabolic Engineering ◽  
Clostridium Acetobutylicum ◽  
Functional Characterization ◽  
Scale Model ◽  
Primary Metabolism ◽  
Scale Analysis ◽  
Content Type ◽  
Commercial Process

ABSTRACTEngineering industrial microorganisms for ambitious applications, for example, the production of second-generation biofuels such as butanol, is impeded by a lack of knowledge of primary metabolism and its regulation. A quantitative system-scale analysis was applied to the biofuel-producing bacteriumClostridium acetobutylicum, a microorganism used for the industrial production of solvent. An improved genome-scale model,iCac967, was first developed based on thorough biochemical characterizations of 15 key metabolic enzymes and on extensive literature analysis to acquire accurate fluxomic data. In parallel, quantitative transcriptomic and proteomic analyses were performed to assess the number of mRNA molecules per cell for all genes under acidogenic, solventogenic, and alcohologenic steady-state conditions as well as the number of cytosolic protein molecules per cell for approximately 700 genes under at least one of the three steady-state conditions. A complete fluxomic, transcriptomic, and proteomic analysis applied to different metabolic states allowed us to better understand the regulation of primary metabolism. Moreover, this analysis enabled the functional characterization of numerous enzymes involved in primary metabolism, including (i) the enzymes involved in the two different butanol pathways and their cofactor specificities, (ii) the primary hydrogenase and its redox partner, (iii) the major butyryl coenzyme A (butyryl-CoA) dehydrogenase, and (iv) the major glyceraldehyde-3-phosphate dehydrogenase. This study provides important information for further metabolic engineering ofC. acetobutylicumto develop a commercial process for the production ofn-butanol.IMPORTANCECurrently, there is a resurgence of interest inClostridium acetobutylicum, the biocatalyst of the historical Weizmann process, to producen-butanol for use both as a bulk chemical and as a renewable alternative transportation fuel. To develop a commercial process for the production ofn-butanol via a metabolic engineering approach, it is necessary to better characterize both the primary metabolism ofC. acetobutylicumand its regulation. Here, we apply a quantitative system-scale analysis to acidogenic, solventogenic, and alcohologenic steady-stateC. acetobutylicumcells and report for the first time quantitative transcriptomic, proteomic, and fluxomic data. This approach allows for a better understanding of the regulation of primary metabolism and for the functional characterization of numerous enzymes involved in primary metabolism.

scholarly journals d-2,3-Butanediol Production Due to Heterologous Expression of an Acetoin Reductase in Clostridium acetobutylicum

2011 ◽  
Vol 77 (8) ◽  
pp. 2582-2588 ◽  
Author(s):  
Marco A. J. Siemerink ◽  
Wouter Kuit ◽  
Ana M. López Contreras ◽  
Gerrit Eggink ◽  
John van der Oost ◽  
...  
Keyword(s):  
Experimental Data ◽  
Heterologous Expression ◽  
Metabolic Network ◽  
Clostridium Acetobutylicum ◽  
Clostridium Beijerinckii ◽  
Racemic Mixture ◽  
Gene Encoding ◽  
Content Type ◽  
Batch Cultures

ABSTRACTAcetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions,Clostridium acetobutylicumATCC 824 (and strains derived from it) generates bothd- andl-stereoisomers of acetoin, but because of the absence of an ACR enzyme, it does not produce 2,3-butanediol. A gene encoding ACR fromClostridium beijerinckiiNCIMB 8052 was functionally expressed inC. acetobutylicumunder the control of two strong promoters, the constitutivethlpromoter and the late exponentialadcpromoter. Both ACR-overproducing strains were grown in batch cultures, during which 89 to 90% of the natively produced acetoin was converted to 20 to 22 mMd-2,3-butanediol. The addition of a racemic mixture of acetoin led to the production of bothd-2,3-butanediol andmeso-2,3-butanediol. A metabolic network that is in agreement with the experimental data is proposed. Native 2,3-butanediol production is a first step toward a potential homofermentative 2-butanol-producing strain ofC. acetobutylicum.

scholarly journals Enhanced Butanol Production Obtained by Reinforcing the Direct Butanol-Forming Route in Clostridium acetobutylicum

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Yu-Sin Jang ◽  
Jin Young Lee ◽  
Joungmin Lee ◽  
Jin Hwan Park ◽  
Jung Ae Im ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Clostridium Acetobutylicum ◽  
Metabolic Flux ◽  
Genetic Manipulation ◽  
High Titer ◽  
Strain Improvement ◽  
High Yield ◽  
Butanol Production ◽  
Wild Type ◽  
Content Type

ABSTRACTButanol is an important industrial solvent and advanced biofuel that can be produced by biphasic fermentation byClostridium acetobutylicum. It has been known that acetate and butyrate first formed during the acidogenic phase are reassimilated to form acetone-butanol-ethanol (cold channel). Butanol can also be formed directly from acetyl-coenzyme A (CoA) through butyryl-CoA (hot channel). However, little is known about the relative contributions of the two butanol-forming pathways. Here we report that the direct butanol-forming pathway is a better channel to optimize for butanol production through metabolic flux and mass balance analyses. Butanol production through the hot channel was maximized by simultaneous disruption of theptaandbukgenes, encoding phosphotransacetylase and butyrate kinase, while theadhE1D485Ggene, encoding a mutated aldehyde/alcohol dehydrogenase, was overexpressed. The ratio of butanol produced through the hot channel to that produced through the cold channel increased from 2.0 in the wild type to 18.8 in the engineered BEKW(pPthlAAD**) strain. By reinforcing the direct butanol-forming flux inC. acetobutylicum, 18.9 g/liter of butanol was produced, with a yield of 0.71 mol butanol/mol glucose by batch fermentation, levels which are 160% and 245% higher than those obtained with the wild type. By fed-batch culture of this engineered strain within siturecovery, 585.3 g of butanol was produced from 1,861.9 g of glucose, with the yield of 0.76 mol butanol/mol glucose and productivity of 1.32 g/liter/h. Studies of two butanol-forming routes and their effects on butanol production inC. acetobutylicumdescribed here will serve as a basis for further metabolic engineering of clostridia aimed toward developing a superior butanol producer.IMPORTANCERenewable biofuel is one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has the potential to replace gasoline. Due to the lack of efficient genetic manipulation tools, however, strain improvement has been rather slow. Furthermore, complex metabolic characteristics of acidogenesis followed by solventogenesis in this strain have hampered development of engineered clostridia having highly efficient and selective butanol production capability. Here we report for the first time the results of systems metabolic engineering studies of two butanol-forming routes and their relative importances in butanol production. Based on these findings, a metabolically engineeredClostridium acetobutylicumstrain capable of producing butanol to a high titer with high yield and selectivity could be developed by reinforcing the direct butanol-forming flux.

scholarly journals Butanol Production from Crystalline Cellulose by Cocultured Clostridium thermocellum and Clostridium saccharoperbutylacetonicum N1-4

2011 ◽  
Vol 77 (18) ◽  
pp. 6470-6475 ◽  
Author(s):  
Shunichi Nakayama ◽  
Keiji Kiyoshi ◽  
Toshimori Kadokura ◽  
Atsumi Nakazato
Keyword(s):  
Clostridium Acetobutylicum ◽  
Clostridium Thermocellum ◽  
Clostridium Beijerinckii ◽  
Crystalline Cellulose ◽  
Butanol Production ◽  
Content Type ◽  
Coculture System

ABSTRACTWe investigated butanol production from crystalline cellulose by cocultured cellulolyticClostridium thermocellumand the butanol-producing strain,Clostridium saccharoperbutylacetonicum(strain N1-4). Butanol was produced from Avicel cellulose after it was incubated withC. thermocellumfor at least 24 h at 60°C before the addition of strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by coculturedClostridium acetobutylicumandClostridium beijerinckiithan by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this coculture system.

scholarly journals Arabinose-Induced Catabolite Repression as a Mechanism for Pentose Hierarchy Control inClostridium acetobutylicumATCC 824

mSystems ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Matthew D. Servinsky ◽  
Rebecca L. Renberg ◽  
Matthew A. Perisin ◽  
Elliot S. Gerlach ◽  
Sanchao Liu ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Genetic Engineering ◽  
Catabolite Repression ◽  
Clostridium Acetobutylicum ◽  
Regulation Mechanism ◽  
Mrna Levels ◽  
Chemical Production ◽  
Content Type ◽  
Commodity Chemicals ◽  
Pentose Utilization

ABSTRACTBacterial fermentation of carbohydrates from sustainable lignocellulosic biomass into commodity chemicals by the anaerobic bacteriumClostridium acetobutylicumis a promising alternative source to fossil fuel-derived chemicals. Recently, it was demonstrated that xylose is not appreciably fermented in the presence of arabinose, revealing a hierarchy of pentose utilization in this organism (L. Aristilde, I. A. Lewis, J. O. Park, and J. D. Rabinowitz, Appl Environ Microbiol 81:1452–1462, 2015,https://doi.org/10.1128/AEM.03199-14). The goal of the current study is to characterize the transcriptional regulation that occurs and perhaps drives this pentose hierarchy. Carbohydrate consumption rates showed that arabinose, like glucose, actively represses xylose utilization in cultures fermenting xylose. Further, arabinose addition to xylose cultures led to increased acetate-to-butyrate ratios, which indicated a transition of pentose catabolism from the pentose phosphate pathway to the phosphoketolase pathway. Transcriptome sequencing (RNA-Seq) confirmed that arabinose addition to cells actively growing on xylose resulted in increased phosphoketolase (CA_C1343) mRNA levels, providing additional evidence that arabinose induces this metabolic switch. A significant overlap in differentially regulated genes after addition of arabinose or glucose suggested a common regulation mechanism. A putative open reading frame (ORF) encoding a potential catabolite repression phosphocarrier histidine protein (Crh) was identified that likely participates in the observed transcriptional regulation. These results substantiate the claim that arabinose is utilized preferentially over xylose inC. acetobutylicumand suggest that arabinose can activate carbon catabolite repression via Crh. Furthermore, they provide valuable insights into potential mechanisms for altering pentose utilization to modulate fermentation products for chemical production.IMPORTANCEClostridium acetobutylicumcan ferment a wide variety of carbohydrates to the commodity chemicals acetone, butanol, and ethanol. Recent advances in genetic engineering have expanded the chemical production repertoire ofC. acetobutylicumusing synthetic biology. Due to its natural properties and genetic engineering potential, this organism is a promising candidate for converting biomass-derived feedstocks containing carbohydrate mixtures to commodity chemicals via natural or engineered pathways. Understanding how this organism regulates its metabolism during growth on carbohydrate mixtures is imperative to enable control of synthetic gene circuits in order to optimize chemical production. The work presented here unveils a novel mechanism via transcriptional regulation by a predicted Crh that controls the hierarchy of carbohydrate utilization and is essential for guiding robust genetic engineering strategies for chemical production.

scholarly journals Engineered Synthetic Pathway for Isopropanol Production in Escherichia coli

2007 ◽  
Vol 73 (24) ◽  
pp. 7814-7818 ◽  
Author(s):  
T. Hanai ◽  
S. Atsumi ◽  
J. C. Liao
Keyword(s):  
Escherichia Coli ◽  
Secondary Alcohol ◽  
Clostridium Beijerinckii ◽  
Synthetic Pathway ◽  
E Coli ◽  
Coa Transferase ◽  
Secondary Alcohol Dehydrogenase ◽  
Acetoacetate Decarboxylase ◽  
Shake Flasks ◽  
K 12

ABSTRACT A synthetic pathway was engineered in Escherichia coli to produce isopropanol by expressing various combinations of genes from Clostridium acetobutylicum ATCC 824, E. coli K-12 MG1655, Clostridium beijerinckii NRRL B593, and Thermoanaerobacter brockii HTD4. The strain with the combination of C. acetobutylicum thl (acetyl-coenzyme A [CoA] acetyltransferase), E. coli atoAD (acetoacetyl-CoA transferase), C. acetobutylicum adc (acetoacetate decarboxylase), and C. beijerinckii adh (secondary alcohol dehydrogenase) achieved the highest titer. This strain produced 81.6 mM isopropanol in shake flasks with a yield of 43.5% (mol/mol) in the production phase. To our knowledge, this work is the first to produce isopropanol in E. coli, and the titer exceeded that from the native producers.

scholarly journals βγ-Crystallination Endows a Novel Bacterial Glycoside Hydrolase 64 with Ca2+-Dependent Activity Modulation

2019 ◽  
Vol 201 (23) ◽  
Author(s):  
Bal Krishnan ◽  
Shanti Swaroop Srivastava ◽  
Venu Sankeshi ◽  
Rupsi Garg ◽  
Sudhakar Srivastava ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Domain Architecture ◽  
Enzyme Reaction ◽  
Structural Features ◽  
Clostridium Beijerinckii ◽  
Biofuel Production ◽  
Carbohydrate Binding ◽  
Reaction Products ◽  
Vast Number ◽  
Content Type

ABSTRACT The prokaryotic βγ-crystallins are a large group of uncharacterized domains with Ca2+-binding motifs. We have observed that a vast number of these domains are found appended to other domains, in particular, the carbohydrate-active enzyme (CAZy) domains. To elucidate the functional significance of these prospective Ca2+ sensors in bacteria and this widespread domain association, we have studied one typical example from Clostridium beijerinckii, a bacterium known for its ability to produce acetone, butanol, and ethanol through fermentation of several carbohydrates. This novel glycoside hydrolase of family 64 (GH64), which we named glucanallin, is composed of a βγ-crystallin domain, a GH64 domain, and a carbohydrate-binding module 56 (CBM56). The substrates of GH64, β-1,3-glucans, are the targets for industrial biofuel production due to their plenitude. We have examined the Ca2+-binding properties of this protein, assayed its enzymatic activity, and analyzed the structural features of the β-1,3-glucanase domain through its high-resolution crystal structure. The reaction products resulting from the enzyme reaction of glucanallin reinforce the mixed nature of GH64 enzymes, in contrast to the prevailing notion of them being an exotype. Upon disabling Ca2+ binding and comparing different domain combinations, we demonstrate that the βγ-crystallin domain in glucanallin acts as a Ca2+ sensor and enhances the glycolytic activity of glucanallin through Ca2+ binding. We also compare the structural peculiarities of this new member of the GH64 family to two previously studied members. IMPORTANCE We have biochemically and structurally characterized a novel glucanase from the less studied GH64 family in a bacterium significant for fermentation of carbohydrates into biofuels. This enzyme displays a peculiar property of being distally modulated by Ca2+ via assistance from a neighboring βγ-crystallin domain, likely through changes in the domain interface. In addition, this enzyme is found to be optimized for functioning in an acidic environment, which is in line with the possibility of its involvement in biofuel production. Multiple occurrences of a similar domain architecture suggest that such a “βγ-crystallination”-mediated Ca2+ sensitivity may be widespread among bacterial proteins.

scholarly journals Reconstruction of an Acetogenic 2,3-Butanediol Pathway Involving a Novel NADPH-Dependent Primary-Secondary Alcohol Dehydrogenase

2014 ◽  
Vol 80 (11) ◽  
pp. 3394-3403 ◽  
Author(s):  
Michael Köpke ◽  
Monica L. Gerth ◽  
Danielle J. Maddock ◽  
Alexander P. Mueller ◽  
FungMin Liew ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Secondary Alcohol ◽  
Acetolactate Synthase ◽  
Chemical Production ◽  
Content Type ◽  
E Coli ◽  
Gas Fermentation ◽  
Secondary Alcohol Dehydrogenase ◽  
Clostridium Autoethanogenum ◽  
Butanediol Dehydrogenase

ABSTRACTAcetogenic bacteria use CO and/or CO2plus H2as their sole carbon and energy sources. Fermentation processes with these organisms hold promise for producing chemicals and biofuels from abundant waste gas feedstocks while simultaneously reducing industrial greenhouse gas emissions. The acetogenClostridium autoethanogenumis known to synthesize the pyruvate-derived metabolites lactate and 2,3-butanediol during gas fermentation. Industrially, 2,3-butanediol is valuable for chemical production. Here we identify and characterize theC. autoethanogenumenzymes for lactate and 2,3-butanediol biosynthesis. The putativeC. autoethanogenumlactate dehydrogenase was active when expressed inEscherichia coli. The 2,3-butanediol pathway was reconstituted inE. coliby cloning and expressing the candidate genes for acetolactate synthase, acetolactate decarboxylase, and 2,3-butanediol dehydrogenase. Under anaerobic conditions, the resultingE. colistrain produced 1.1 ± 0.2 mM 2R,3R-butanediol (23 μM h−1optical density unit−1), which is comparable to the level produced byC. autoethanogenumduring growth on CO-containing waste gases. In addition to the 2,3-butanediol dehydrogenase, we identified a strictly NADPH-dependent primary-secondary alcohol dehydrogenase (CaADH) that could reduce acetoin to 2,3-butanediol. Detailed kinetic analysis revealed that CaADH accepts a range of 2-, 3-, and 4-carbon substrates, including the nonphysiological ketones acetone and butanone. The high activity of CaADH toward acetone led us to predict, and confirm experimentally, thatC. autoethanogenumcan act as a whole-cell biocatalyst for converting exogenous acetone to isopropanol. Together, our results functionally validate the 2,3-butanediol pathway fromC. autoethanogenum, identify CaADH as a target for further engineering, and demonstrate the potential ofC. autoethanogenumas a platform for sustainable chemical production.

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