A novel bifunctional aldehyde/alcohol dehydrogenase mediating ethanol formation from acetyl-CoA in hyperthermophiles

2020 ◽  
Author(s):  
Qiang Wang ◽  
Chong Sha ◽  
Hongcheng Wang ◽  
Kesen Ma ◽  
Juergen Wiegel ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Alcohol Dehydrogenase ◽  
Ethanol Production ◽  
Ethanol Fermentation ◽  
Cellulosic Ethanol ◽  
Decarboxylase Activity ◽  
Aldh Activity ◽  
Acetyl Coa ◽  
Ethanol Formation

Abstract Background: Hyperthermophilic fermentation at temperatures above 80 °C allows in situ product removal to mitigate the ethanol toxicity, and reduces microbial contamination without autoclaving/cooling of feedstock. Many species of Thermotoga grow at temperatures up to 90 °C, and have enzymes to degrade and utilize lignocelluloses, which provide advantages for achieving consolidated processes of cellulosic ethanol production. However, no CoA-dependent aldehyde dehydrogenase (CoA-Aldh) from any hyperthermophiles has been documented in literature so far. The pyruvate ferredoxin oxidoreductases from hyperthermophiles have pyruvate decarboxylase activity, which convert about 2% and 98% of pyruvate to acetaldehyde and acetyl-CoA (ac-CoA), respectively. Acetyl-CoA can be converted to acetic acid, if there is no CoA-Aldh to convert ac-CoA to acetaldehyde and further to ethanol. Therefore, the current study aimed to identify and characterize a CoA-Aldh activity that mediates ethanol fermentation in hyperthermophiles.Results: In Thermotoga neapolitana (Tne), a hyperthermophilic iron-acetaldehyde/alcohol dehydrogenase (Fe-AAdh) was, for the first time, revealed to catalyze the ac-CoA reduction to form ethanol via an acetaldehyde intermediate, while the annotated aldh gene in Tne genome only encodes a CoA-independent Aldh that oxidizes aldehyde to acetic acid. Three other Tne alcohol dehydrogenases (Adh) exhibited specific physiological roles in ethanol formation and consumption: Fe-Adh2 mainly catalyzed the reduction of acetaldehyde to produce ethanol, and Fe-Adh1 showed significant activities only under extreme conditions, while Zn-Adh showed special activity in ethanol oxidation. In the in vitro formation of ethanol from ac-CoA, a strong synergy was observed between Fe-Adh1 and Fe-AAdh. The Fe-AAdh gene is highly conserved in Thermotoga spp. and in Pyrococus sp., which is probably responsible for ethanol metabolism in hyperthermophiles.Conclusions: Hyperthermophilic Thermotoga spp. are excellent candidates for biosynthesis of cellulosic ethanol fermentation strains. The finding of a novel hyperthermophilic CoA-Aldh activity of Tne Fe-AAdh revealed the existence of a hyperthermophilic fermentation pathway from ac-CoA to ethanol, which offers a basic frame for in vitro synthesis of a highly active AAdh for effective ethanol fermentation pathway in hyperthermophiles, which is a key element for the approach to the consolidated processes of cellulosic ethanol production.

scholarly journals Engineering Pichia pastoris with surface-display minicellulosomes for carboxymethyl cellulose hydrolysis and ethanol production

2020 ◽  
Author(s):  
Ce Dong ◽  
Jie Qiao ◽  
Xinping Wang ◽  
Wenli Sun ◽  
Lixia Chen ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Ethanol Production ◽  
Carboxymethyl Cellulose ◽  
Cellulosic Ethanol ◽  
Consolidated Bioprocessing ◽  
Surface Display ◽  
Cell Factory ◽  
Carbohydrate Binding ◽  
Surface Assembly

Abstract Backgrounds: Engineering yeast as a consolidated bioprocessing (CBP) microorganism by surface assembly of cellulosomes has been aggressively utilized for cellulosic ethanol production. However, most of the previous studies focused on Saccharomyces cerevisiae, achieving efficient conversion of phosphoric acid-swollen cellulose (PASC) or microcrystalline cellulose (Avicel) but not carboxymethyl cellulose (CMC) to ethanol, with an average titer below 2 g/L. Results: Harnessing an ultra-high-affinity IM7/CL7 protein pair, here we describe a method to engineer Pichia pastoris with minicellulosomes by in vitro assembly of three recombinant cellulases including an endoglucanase (EG), an exoglucanase (CBH) and a β-glucosidase (BGL), as well as a carbohydrate binding module (CBM) on the cell surface. For the first time, the engineered yeasts enable efficient and direct conversion of CMC to bioethanol, observing an impressive ethanol titer of 5.1 g/L. Conclusions: The research promotes the application of P. pastoris as a CBP cell factory in cellulosic ethanol production and provides a promising platform for screening the cellulases from different species to construct surface-assembly celluosome.

scholarly journals Pyruvate-to-ethanol pathway in Entamoeba histolytica

1978 ◽  
Vol 171 (1) ◽  
pp. 225-230 ◽  
Author(s):  
H S Lo ◽  
R E Reeves
Keyword(s):  
Enzyme Activity ◽  
Alcohol Dehydrogenase ◽  
Ethanol Production ◽  
Entamoeba Histolytica ◽  
Aldehyde Dehydrogenase ◽  
Pyruvate Decarboxylase ◽  
Acetyl Coa ◽  
Key Enzyme ◽  
Pyruvate Synthase ◽  
Soluble Enzymes

The pyruvate-to-ethanol pathway in Entamoeba histolytica is unusual when compared with most investigated organisms. Pyruvate decarboxylase (EC 4.1.1.1), a key enzyme for ethanol production, is not found. Pyruvate is converted into acetyl-CoA and CO2 by the enzyme pyruvate synthase (EC 1.2.7.1), which has been demonstrated previously in this parasitic amoeba. Acetyl-CoA is reduced to acetaldehyde and CoA by the enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10) at an enzyme activity of 9 units per g of fresh cells with NADH as a reductant. Acetaldehyde is further reduced by either a previously identified NADP+-linked alcohol dehydrogenase or by a newly found NAD+-linked alcohol dehydrogenase at an enzyme activity of 136 units per g of fresh cells. Ethanol is identified as the product of soluble enzymes of amoeba acting on pyruvate or acetyl-CoA. This result is confirmed by radioactive isotopic, spectrophotometric and gas-chromatographic methods.

scholarly journals A novel bifunctional aldehyde/alcohol dehydrogenase catalyzing reduction of acetyl-CoA to ethanol at temperatures up to 95 °C

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Qiang Wang ◽  
Chong Sha ◽  
Hongcheng Wang ◽  
Kesen Ma ◽  
Juergen Wiegle ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Alcohol Dehydrogenase ◽  
Aldehyde Dehydrogenase ◽  
Half Life ◽  
Cellulosic Ethanol ◽  
Pyrococcus Furiosus ◽  
Biochemical Properties ◽  
Cellulosic Biomass ◽  
Acetyl Coenzyme A ◽  
First Time

AbstractHyperthermophilic Thermotoga spp. are excellent candidates for the biosynthesis of cellulosic ethanol producing strains because they can grow optimally at 80 °C with ability to degrade and utilize cellulosic biomass. In T. neapolitana (Tne), a putative iron-containing alcohol dehydrogenase was, for the first time, revealed to be a bifunctional aldehyde/alcohol dehydrogenase (Fe-AAdh) that catalyzed both reactions from acetyl-coenzyme A (ac-CoA) to acetaldehyde (ac-ald), and from ac-ald to ethanol, while the putative aldehyde dehydrogenase (Aldh) exhibited only CoA-independent activity that oxidizes ac-ald to acetic acid. The biochemical properties of Fe-AAdh were characterized, and bioinformatics were analyzed. Fe-AAdh exhibited the highest activities for the reductions of ac-CoA and acetaldehyde at 80–85 °C, pH 7.54, and had a 1-h half-life at about 92 °C. The Fe-AAdh gene is highly conserved in Thermotoga spp., Pyrococcus furiosus and Thermococcus kodakarensis, indicating the existence of a fermentation pathway from ac-CoA to ethanol via acetaldehyde as the intermediate in hyperthermophiles.

Transgenic cotton (Gossypium hirsutum) over-expressing alcohol dehydrogenase shows increased ethanol fermentation but no increase in tolerance to oxygen deficiency

2000 ◽  
Vol 27 (11) ◽  
pp. 1041 ◽  
Author(s):  
Marc H. Ellis ◽  
Anthony A. Millar ◽  
Danny J. Llewellyn ◽  
W. James Peacock ◽  
Elizabeth S. Dennis
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Fermentation ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Mild Stress ◽  
Waterlogging Tolerance ◽  
Over Expression ◽  
Camv 35S ◽  
Adh Activity

Cotton (Gossypium hirsutumL.) was transformed with constructs for the over-expression of two enzymes involved in ethanol fermentation, alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC), with the goal of increasing waterlogging tolerance. Four independent transgenic lines transformed with the cotton Adh2 cDNA driven by the CaMV 35S promoter showed ectopic expression of this isozyme in leaves and up to 20-fold greater in vitro ADH activity in roots. Under conditions of O2 deficiency, excised roots from these transgenic plants showed up to 80% increase in ethanol evolution compared to untransformed roots. Conversely, one line transformed with a construct containing the Adh2 coding region in the antisense orientation showed a 65% decrease in ADH activity and a 25% decrease in ethanol production from anaerobic roots relative to untransformed cotton. Lines transformed with a rice Pdc1 cDNA driven by the CaMV 35S promoter showed high levels of expression of the transgene-encoded protein in leaves, but only very low levels of protein and no in vitro enzyme activity detectable in the roots of these plants. Roots from plants transformed with the 35S-Pdc construct did not produce more ethanol than roots from untransformed controls. We tested the ability of cotton roots to withstand low O2 treatments under hydroponic conditions. Neither the ‘ADH’ nor the ‘PDC’ transgenics showed more tolerance than the wild-type on the basis of root growth under a mild stress (5% O2), a strong stress (0% O2 with or without a 5% O2 pretreatment), or in recovery growth following these treatments. Our results show that over-expression of ADH can lead to ethanol over-production (even though the activity of this enzyme by far exceeds that of PDC, its precursor in the pathway), but this is not sufficient to increase waterlogging tolerance in cotton.

scholarly journals The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene,adhE, Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

2015 ◽  
Vol 197 (8) ◽  
pp. 1386-1393 ◽  
Author(s):  
Jonathan Lo ◽  
Tianyong Zheng ◽  
Shuen Hon ◽  
Daniel G. Olson ◽  
Lee R. Lynd
Keyword(s):  
Ethanol Production ◽  
Aldehyde Dehydrogenase ◽  
Clostridium Thermocellum ◽  
Deletion Strain ◽  
Aldh Activity ◽  
Content Type ◽  
Ethanol Formation ◽  
Cell Extracts ◽  
Thermoanaerobacterium Saccharolyticum ◽  
Dehydrogenase Gene

ABSTRACTThermoanaerobacterium saccharolyticumandClostridium thermocellumare anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene,adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of theadhEgene inC. thermocellumandT. saccharolyticum. Deletion ofadhEreduced ethanol production by >95% in bothT. saccharolyticumandC. thermocellum, confirming thatadhEis necessary for ethanol formation in both organisms. In bothadhEdeletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. InT. saccharolyticum, theadhEdeletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in theT. saccharolyticumparent strain but did not increase activity in theadhEdeletion strain, suggesting that ALDH activity was inhibited. InC. thermocellum, theadhEdeletion strain lost >90% of ALDH and ADH activity in cell extracts. TheC. thermocellumadhEdeletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase.IMPORTANCEThermoanaerobacterium saccharolyticumandClostridium thermocellumare bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol dehydrogenases which could be responsible for alcohol formation. This study explores the inactivation ofadhE, a gene encoding a bifunctional alcohol and aldehyde dehydrogenase. Deletion ofadhEreduced ethanol production by >95% in bothT. saccharolyticumandC. thermocellum, confirming thatadhEis necessary for ethanol formation in both organisms. In strains withoutadhE, we note changes in biochemical activity, product formation, and growth.

scholarly journals Thermotolerant Zymomonas mobilis: Comparison of Ethanol Fermentation Capability with that of an Efficient Type Strain

2007 ◽  
Vol 1 (1) ◽  
pp. 59-65 ◽  
Author(s):  
Kaewta Sootsuwan ◽  
Akira Irie ◽  
Masayuki Murata ◽  
Noppon Lertwattanasakul ◽  
Pornthap Thanonkeo ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Ethanol Fermentation ◽  
Zymomonas Mobilis ◽  
Pyruvate Decarboxylase ◽  
Total Activity ◽  
Transcriptional Level ◽  
Ethanol Formation ◽  
Different Temperatures ◽  
Efficient Type ◽  
Better Than

Zymomonas mobilis is an alternative microorganism to Saccharomyces cerevisiae for ethanol production. To find a thermotolerant Z. mobilis strain, the growth and ethanol production of four isolates in Thailand were compared with those of the efficient strain ZM4 (NRRL B-14023) at different temperatures. One of the selected strains, TISTR 405, was found to grow and produce ethanol even at 39°C to an extent similar to that at 30°C, and the growth and ethanol productivity at 39°C were better than those of ZM4 at 30°C, suggesting that TISTR 405 is suitable for ethanol fermentation at high temperatures. Analysis of genes directly related to ethanol formation or degradation, adhA, adhB and pdc, encoding alcohol dehydrogenase (Adh) A, AdhB and pyruvate decarboxylase, respectively, revealed that these genes were highly conserved in both strains. Comparison of their gene expression and activity of the products in both TISTR 405 and ZM4 at different temperatures or growth phases indicated that there was not a great difference at the transcriptional level, but the total activity of AdhA and AdhB in TISTR 405 was higher than that in ZM4. Both strains showed a significant increase in AdhB activity in the stationary phase.

scholarly journals Energy Conservation Associated with Ethanol Formation from H2and CO2in Clostridium autoethanogenum Involving Electron Bifurcation

2015 ◽  
Vol 197 (18) ◽  
pp. 2965-2980 ◽  
Author(s):  
Johanna Mock ◽  
Yanning Zheng ◽  
Alexander P. Mueller ◽  
San Ly ◽  
Loan Tran ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Acetyl Coa ◽  
Content Type ◽  
Substrate Level Phosphorylation ◽  
Clostridium Ljungdahlii ◽  
Ethanol Formation ◽  
Cell Extracts ◽  
Substrate Level ◽  
Clostridium Autoethanogenum ◽  
Specific Activities

ABSTRACTMost acetogens can reduce CO2with H2to acetic acid via the Wood-Ljungdahl pathway, in which the ATP required for formate activation is regenerated in the acetate kinase reaction. However, a few acetogens, such asClostridium autoethanogenum,Clostridium ljungdahlii, andClostridium ragsdalei, also form large amounts of ethanol from CO2and H2. How these anaerobes with a growth pH optimum near 5 conserve energy has remained elusive. We investigated this question by determining the specific activities and cofactor specificities of all relevant oxidoreductases in cell extracts of H2/CO2-grownC. autoethanogenum. The activity studies were backed up by transcriptional and mutational analyses. Most notably, despite the presence of six hydrogenase systems of various types encoded in the genome, the cells appear to contain only one active hydrogenase. The active [FeFe]-hydrogenase is electron bifurcating, with ferredoxin and NADP as the two electron acceptors. Consistently, most of the other active oxidoreductases rely on either reduced ferredoxin and/or NADPH as the electron donor. An exception is ethanol dehydrogenase, which was found to be NAD specific. Methylenetetrahydrofolate reductase activity could only be demonstrated with artificial electron donors. Key to the understanding of this energy metabolism is the presence of membrane-associated reduced ferredoxin:NAD+oxidoreductase (Rnf), of electron-bifurcating and ferredoxin-dependent transhydrogenase (Nfn), and of acetaldehyde:ferredoxin oxidoreductase, which is present with very high specific activities in H2/CO2-grown cells. Based on these findings and on thermodynamic considerations, we propose metabolic schemes that allow, depending on the H2partial pressure, the chemiosmotic synthesis of 0.14 to 1.5 mol ATP per mol ethanol synthesized from CO2and H2.IMPORTANCEEthanol formation from syngas (H2, CO, and CO2) and from H2and CO2that is catalyzed by bacteria is presently a much-discussed process for sustainable production of biofuels. Although the process is already in use, its biochemistry is only incompletely understood. The most pertinent question is how the bacteria conserve energy for growth during ethanol formation from H2and CO2, considering that acetyl coenzyme A (acetyl-CoA), is an intermediate. Can reduction of the activated acetic acid to ethanol with H2be coupled with the phosphorylation of ADP? Evidence is presented that this is indeed possible, via both substrate-level phosphorylation and electron transport phosphorylation. In the case of substrate-level phosphorylation, acetyl-CoA reduction to ethanol proceeds via free acetic acid involving acetaldehyde:ferredoxin oxidoreductase (carboxylate reductase).

scholarly journals Ferredoxin:NAD+Oxidoreductase of Thermoanaerobacterium saccharolyticum and Its Role in Ethanol Formation

2016 ◽  
Vol 82 (24) ◽  
pp. 7134-7141 ◽  
Author(s):  
Liang Tian ◽  
Jonathan Lo ◽  
Xiongjun Shao ◽  
Tianyong Zheng ◽  
Daniel G. Olson ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Redox Balance ◽  
Wild Type ◽  
Anaerobic Microorganism ◽  
Content Type ◽  
Ethanol Formation ◽  
Thermoanaerobacterium Saccharolyticum ◽  
Different Strains ◽  
Electron Carriers

ABSTRACTFerredoxin:NAD+oxidoreductase (NADH-FNOR) catalyzes the transfer of electrons from reduced ferredoxin to NAD+. This enzyme has been hypothesized to be the main enzyme responsible for ferredoxin oxidization in the NADH-based ethanol pathway inThermoanaerobacterium saccharolyticum; however, the corresponding gene has not yet been identified. Here, we identified the Tsac_1705 protein as a candidate FNOR based on the homology of its functional domains. We then confirmed its activityin vitrowith a ferredoxin-based FNOR assay. To determine its role in metabolism, thetsac_1705gene was deleted in different strains ofT. saccharolyticum. In wild-typeT. saccharolyticum, deletion oftsac_1705resulted in a 75% loss of NADH-FNOR activity, which indicated that Tsac_1705 is the main NADH-FNOR inT.saccharolyticum. When both NADH- and NADPH-linked FNOR genes were deleted, the ethanol titer decreased and the ratio of ethanol to acetate approached unity, indicative of the absence of FNOR activity. Finally, we tested the effect of heterologous expression of Tsac_1705 inClostridium thermocellumand found improvements in both the titer and the yield of ethanol.IMPORTANCERedox balance plays a crucial role in many metabolic engineering strategies. Ferredoxins are widely used as electron carriers for anaerobic microorganism and plants. This study identified the gene responsible for electron transfer from ferredoxin to NAD+, a key reaction in the ethanol production pathway of this organism and many other metabolic pathways. Identification of this gene is an important step in transferring the ethanol production ability of this organism to other organisms.

Callus Induction and Plant Regeneration in Rauvolfia Serpentina (L.) Benth Ex. Kurz.

2009 ◽  
Vol 24 ◽  
pp. 82-88 ◽  
Author(s):  
Saraswoti Aryal ◽  
Sanu Devi Joshi
Keyword(s):  
Acetic Acid ◽  
Callus Induction ◽  
Coconut Milk ◽  
Ms Medium ◽  
Rauvolfia Serpentina ◽  
History Museum ◽  
Short Period ◽  
Murashige Skoog ◽  
Callus Tissue Culture

Rauvolfia serpentina (L.) ex. Kurz is an important medicinal plant. Callus induction and regeneration was studied from stem explant of in-vitro grown plant of Rauvolfia serpentina(L.) Benth. ex Kurz (Apocynaceae) on Murashige Skoog (1962) medium supplemented with 1mg/l 2,4-Dichlorophenocy acetic acid (2,4-D) and 1mg/l Kinetin (Kn). Vigorous growth of callus occurs after 4 weeks of culture. Callus was sub-cultured on Murashige and Skoog (MS) medium supplemented with different concentration of 2, 4-D (0.5-3.0 mg/l) and 10% coconut milk. Regeneration of plantlets occurred on MS medium containing 3 mg/1 of 2, 4-D and 10% coconut milk. These plantlets were rooted on MS medium supplemented with 1 mg/l IAA .The regenerated plantlets were able to grow on soil after short period ofacclimatization. Key words: Explant; In-vitro culture; MS medium;  2, 4 Dichlorophenoxy acetic acid; Kinetin; Callus; Tissue culture; Coconut milk. Journal of Natural History Museum Vol. 24, 2009 Page: 82-88

Advancement Of Changed Pulsincap Medicate Conveyance Arrangement Of Metronidazole For Tranquilize Focusing

2020 ◽  
Vol 2 (05) ◽  
pp. I-IV
Author(s):  
Roshni Jha ◽  
Anjali Minj
Keyword(s):  
Acetic Acid ◽  
Sodium Alginate ◽  
Guar Gum

A changed Pulsincap measurements type of metronidazole was created to target tranquilize discharge in the colon. Groups of hard gelatin cases were treated with formaldehyde keeping the tops in that capacity. Metronidazole pellets arranged by expulsion spheronization technique were consolidated into these particular container shells and stopped with polymers guar gum, hydroxypropylmethylcellulose 10K, carboxymethylcellulose sodium and sodium alginate independently at fixations 20 mg, 30 mg and 40 mg. The filled cases were totally covered with 5% cellulose acetic acid derivation phthalate to forestall variable gastric purging. All the definitions were tested to decide sedate substance and the capacity of the adjusted Pulsincap to give colon-explicit medication conveyance was surveyed by in vitro tranquilize discharge concentrates in cushion pH 1.2 for 2 h, pH 7.4 (reproduced intestinal liquid) for 3 h and pH 6.8 (animated colonic liquid) for 7 h. The outcomes showed that critical medication discharge happened simply after 5 h from the beginning of analysis.

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