Thermoanaerobacter Ethanolicus Research Articles

Immobilised Alcohol Dehydrogenase and Ene‐Reductase Work in Concert to Reduce Cyclohex‐2‐enone in Bulk Organic Solvent

Enone reduction by ene reductase (ERED) has not been reported yet in bulk organic media probably due to expected challenges with the cofactor recycling using a second enzyme and the exchange of the NAD(P)‐cofactor between the two enzymes. Herein, the combination of an ERED from Zymomonas mobilis (NCR‐ERED) and Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) has been found as an efficient biocatalytic redox system for the reduction of cyclohex‐2‐enone into cyclohexanol in bulk organic solvents. Both enzymes were successfully immobilised on three EziG supports, finding Coral as the most promising carrier for both of them. The highest enzymatic activities were found in hydrophobic solvents such as toluene and isooctane. It was observed that the control of water content and post‐immobilisation treatments enabled retention of enzyme activity. Both enzymes were co‐immobilised on the same bead together with NADP+, leading to the production of a self‐sufficient catalytic system, although the external supplementation of the nicotinamide cofactor was necessary for an efficient enzyme reuse. In this case, no ERED activity loss was detected after two additional cycles, while the ADH experienced less than 20% activity loss upon reuse.

Manipulating Fermentation Pathways in the Hyperthermophilic Archaeon Pyrococcus furiosus for Ethanol Production up to 95°C Driven by Carbon Monoxide Oxidation.

Genetic engineering of hyperthermophilic organisms for the production of fuels and other useful chemicals is an emerging biotechnological opportunity. In particular, for volatile organic compounds such as ethanol, fermentation at high temperatures could allow for straightforward separation by direct distillation. Currently, the upper growth temperature limit for native ethanol producers is 72°C in the bacterium Thermoanaerobacter ethanolicus JW200, and the highest temperature for heterologously-engineered bioethanol production was recently demonstrated at 85°C in the archaeon Pyrococcus furiosus. Here, we describe an engineered strain of P. furiosus that synthesizes ethanol at 95°C, utilizing a homologously-expressed native alcohol dehydrogenase, termed AdhF. Ethanol biosynthesis was compared at 75°C and 95°C with various engineered strains. At lower temperatures, the acetaldehyde substrate for AdhF is most likely produced from acetate by aldehyde ferredoxin oxidoreductase (AOR). At higher temperatures, the effect of AOR on ethanol production is negligible, suggesting that acetaldehyde is produced by pyruvate ferredoxin oxidoreductase (POR) via oxidative decarboxylation of pyruvate, a reaction known to occur only at higher temperatures. Heterologous expression of a carbon monoxide dehydrogenase complex in the AdhF overexpression strain enabled it to use CO as a source of energy, leading to increased ethanol production. A genome reconstruction model for P. furiosus was developed to guide metabolic engineering strategies and understand outcomes. This work opens the door to the potential for 'bioreactive distillation' since fermentation can be performed well above the normal boiling point of ethanol. IMPORTANCE Previously, the highest temperature for biological ethanol production was 85°C. Here, we have engineered ethanol production at 95°C by the hyperthermophilic archaeon Pyrococcus furiosus. Using mutant strains, we showed that ethanol production occurs by different pathways at 75°C and 95°C. In addition, by heterologous expression of a carbon monoxide dehydrogenase complex, ethanol production by this organism was driven by the oxidation of carbon monoxide. A genome reconstruction model for P. furiosus was developed to guide metabolic engineering strategies and understand outcomes.

How Medium Affects the Activity and Stereopreference of Thermoanaerobacter ethanolicus Secondary Alcohol Dehydrogenase Catalyzed Reduction of Ketones

Abstract: The activity and selectivity of W110G Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase were altered by cosubstrate and cosolvent at varying temperatures. A sharp drop in the enantiomeric excess (ee) was observed at 60 oC in the first 3 h, suggesting increased selectivity mistakes in the reduction of 4-phenyl-2-butanone to the expected (S)-4-phenyl-2-butanol using 5% v/v of 2-propanol as a cosubstrate. The ee increased exponentially with cosubstrate concentration, reaching ≥94% with 30-70% v/v 2-propanol. However, a decrease in enzyme activity was noticed at ≥30% v/v 2-propanol by a sharp drop in conversion. The lowest ee (<3%) was registered using 5% v/v 2- propanol at 30-40 oC, which prolonged enzyme life that allowed reversible redox reaction with selectivity mistakes to give the R-alcohol compared to ≥18% ee at 50-60 oC, where faster reaction rates promoted selectivity mistake, but enzyme life was shortened by protein denaturation at the elevated temperatures. Water-miscible and immiscible organic cosolvents (25% v/v) increased enzyme selectivity. For methanol, ethanol, ethylene glycol and tert-butanol, the activity/conversion decreased with an increase in pKa and log P while the stereoselectivity/ee increased.

Heterologous Expression of Thermotolerant α-Glucosidase in Bacillus subtilis 168 and Improving Its Thermal Stability by Constructing Cyclized Proteins

α-glucosidase is an essential enzyme for the production of isomaltooligosaccharides (IMOs). Allowing α-glucosidase to operate at higher temperatures (above 60 °C) has many advantages, including reducing the viscosity of the reaction solution, enhancing the catalytic reaction rate, and achieving continuous production of IMOs. In the present study, the thermal stability of α-glucosidase was significantly improved by constructing cyclized proteins. We screened a thermotolerant α-glucosidase (AGL) with high transglycosylation activity from Thermoanaerobacter ethanolicus JW200 and heterologously expressed it in Bacillus subtilis 168. After forming the cyclized α-glucosidase by different isopeptide bonds (SpyTag/SpyCatcher, SnoopTag/SnoopCatcher, SdyTag/SdyCatcher, RIAD/RIDD), we determined the enzymatic properties of cyclized AGL. The optimal temperature of all cyclized AGL was increased by 5 °C, and their thermal stability was generally improved, with SpyTag-AGL-SpyCatcher having a 1.74-fold increase compared to the wild-type. The results of molecular dynamics simulations showed that the RMSF values of cyclized AGL decreased, indicating that the rigidity of the cyclized protein increased. This study provides an efficient method for improving the thermal stability of α-glucosidase.

Identification, molecular and biochemical characterization of a novel thermoactive and thermostable glucoamylase from Thermoanaerobacter ethanolicus.

We identified a new glucoamylase (TeGA) from Thermoanaerobacter ethanolicus, a thermophilic anaerobic bacterium. Structural studies suggest that TeGA belongs to the family 15 of glycosylhydrolases (GH15). The expression of this enzyme was optimized in E. coli (BL21) cells in order to have the highest amount of soluble protein (around 3mg/l of culture medium). TeGA showed a high optimum temperature of 75°C. It also showed one of the highest specific activities reported for a bacterial glucoamylase (75.3 U/mg) and was also stable in a wide pH range (3.0-10.0). Although the enzyme was preferentially active with maltose, it was also able to hydrolyze different soluble starches such as those from potato, corn or rice. TeGA showed a high thermostability up to around 70°C, which was increased in the presence of PEG8000, and also showed to be stable in the presence of moderate concentrations of ethanol. We propose that TeGA could be suitable for use in different industrial processes such as biofuel production and food processing.

Enhanced catalytic efficiency and thermostability of glucose isomerase from Thermoanaerobacter ethanolicus via site-directed mutagenesis

Glucose isomerase (GI) is a key enzyme in the preparation of high fructose corn syrup (HFCS). In this study, a mutant TEGI-M-L38 M/V137 L (TEGI-M2) of glucose isomerase (TEGI-M) originated from Thermoanaerobacter ethanalicus CCSD1 was obtained by site-directed mutagenesis. The TEGI-M2 showed an optimal activity at 85 ℃ and pH 6.5 with the divalent cations Co2+ and Mg2+. The structural differences between TEGI-M and TEGI-M2 were investigated based on the homology modeling and molecular docking, to elucidate the mechanism of improvement in the enzymatic properties. Compared with the original enzyme, the TEGI-M2 showed a 2.0-fold increased enzyme activity and a decreased Km from 234.2 mM to 85.9 mM. Finally, the application of mutant TEGI-M2 in HFCS one-step biosynthesis was attempted, resulting in a d-fructose yield of 67.3 %, which was 14.3 % higher than that of TEGI-M. This improved catalytic performance of TEGI-M2 was of great importance for the industrial preparation of d-fructose in one-step process.

Single pot biovalorization of food waste to ethanol by Geobacillus and Thermoanaerobacter spp.

The current study was focused on developing a green thermophilic bioprocess to produce bioethanol from the food waste. The stored energy in food waste can be used for bioethanol production that can also help in reducing the land and environment impact of current food waste management processes. Geobacillus thermoglucosidasius was used to produce ethanol from food waste in a single pot at 60 °C. Scaling up the single pot bioprocess from 165ml serum bottles (165ml) to 1L bioreactor increased the ethanol yield from 3.03 g/L to 13 g/L. With scaling up to 1L reactor improved the substrate mass recovery from 39.2% to 92.8%. The ethanol production in 1L was further increased to 16.1g/L by the sequential cultivation of Geobacillus thermoglucosidasius and Thermoanaerobacter ethanolicus. Further, scaling up to 40L reactor gave an ethanol yield of 18.4 g/L and improved the ethanol productivity from 0.07 g/L/h (1L reactor) to 0.15 g/L/h (40L reactor). The sequential cultivation of thermophiles was able to produce ethanol from food waste without pretreatment, giving 70.1L of gasoline equivalent per ton of dry food waste. This is the first report of sequential cultivation of thermophiles for ethanol production using food waste and scaling up to pilot plant.

Enzyme immobilization on porous chitosan hydrogel capsules formed by anionic surfactant gelation.

Sodium dodecyl sulfate (SDS)-chitosan hydrogels have been employed for adsorption of anionic dyes and metallic substances. Two mutant forms of Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) were used as model enzymes to develop a novel enzyme immobilization technique employing newly formulated porous chitosan hydrogels. The enzyme immobilized on chitosan hydrogel capsules formed by 5g/l SDS gelation and subsequent treatment with 0.05M NaOH was 28-35% higher in NADPH production than that formed by 20g/l SDS gelation only under the same conditions. A 48-h asymmetric biphasic reduction of acetophenone with immobilized TeSADH enzyme at 50 °C showed 68% increase in (R)-1-phenylethanol production than the free enzyme. Compared to the free enzyme which denatured and lost its activity at 80 °C, the immobilized enzyme retained about 25% of its initial activity after 2-h incubation. In contrast to the conventional chitosan hydrogel which suffers thermal and operational stability, the newly formulated porous chitosan hydrogel capsules have excellent enzyme loading efficiency and stable at harsh temperatures. Especially, this newly developed enzyme immobilization method would be applicable for food processing.

Characterization of new Baeyer-Villiger monooxygenases for lactonizations in redox-neutral cascades

Technical application of cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 in particular, is hindered by limited enzyme stability. In addition, substrate and product inhibition is a well-known challenge of using CHMO. By site-directed mutagenesis two new combinatorial CHMO variants, CHMO M15 L323C-A325C (M15 DS) and CHMO M16 L323C-A325C (M16 DS), were designed to stabilize the enzyme, by incorporating a reported disulfide bridge into the already published parental CHMO variants: CHMO M15 and CHMO M16. Additionally, the newly described BVMO AFL706 from Aspergillus flavus was characterized for epsilon-caprolactone (ECL) synthesis, for which the enzyme showed significantly higher substrate and product tolerance compared to the wild type. The wild type CHMO and the four variants were applied in a convergent cascade coupled with an alcohol dehydrogenase from Thermoanaerobacter ethanolicus for ECL synthesis. M15 DS and M16 DS variants performed better than the wild type and the parental variants under operational conditions. Subsequently, the cascade was further optimized by means of Design of Experiments, doubling the product yield (21 mM, 27%) with a reduced cofactor amount of 0.5 mM NADP+.

A Hybrid Sequencing Approach Completes the Genome Sequence of Thermoanaerobacter ethanolicus JW 200.

Thermoanaerobacter ethanolicus JW 200 has been identified as a potential sustainable biofuel producer due to its ability to readily ferment carbohydrates to ethanol. A hybrid sequencing approach, combining Oxford Nanopore and Illumina DNA sequence reads, was applied to produce a single contiguous genome sequence of 2,911,280 bp.

Impact of Expanded Small Alkyl-Binding Pocket by Triple Point Mutations on Substrate Specificity of Thermoanaerobacter ethanolicus Secondary Alcohol Dehydrogenase

Site-directed mutagenesis was employed to generate five different triple point mutations in the double mutant (C295A/I86A) of Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) by computer-aided modeling with the aim of widening the small alkyl-binding pocket. TeSADH engineering enables the enzyme to accept sterically hindered substrates that could not be accepted by the wild-type enzyme. The underline in the mutations highlights the additional point mutation on the double mutant TeSADH introduced in this work. The catalytic efficiency (kcat/KM) of the M151A/C295A/I86A triple TeSADH mutant for acetophenone increased about 4.8-fold higher than that of the double mutant. A 2.4-fold increase in conversion of 3'-methylacetophenone to (R)-1-(3-methylphenyl)-ethanol with a yield of 87% was obtained by using V115A/C295A/I86A mutant in asymmetric reduction. The A85G/C295A/I86A mutant also produced (R)-1-(3-methylphenyl)-ethanol (1.7-fold) from 3'-methylacetophenone and (R)-1-(3-methoxyphenyl)-ethanol (1.2-fold) from 3'- methoxyacetophenone, with improved yield. In terms of thermal stability, the M151A/ C295A/I86A and V115A/C295A/I86A mutants significantly increased ΔT1/2 by +6.8°C and +2.4°C, respectively, with thermal deactivation constant (kd) close to the wild-type enzyme. The M151A/C295A/I86A mutant reacts optimally at 70 °C with almost 4 times more residual activity than the wild type. Considering broad substrate tolerance and thermal stability together, it would be promising to produce (R)-1-(3-methylphenyl)-ethanol from 3'- methylacetophenone by V115A/C295A/I86A, and (R)-1-phenylethanol from acetophenone by M151A/C295A/I86A mutant, in large-scale bioreduction processes.

Expression of engineered carbonyl reductase from Ogataea minuta in Rhodococcus opacus and its application to whole-cell bioconversion in anhydrous solvents

The carbonyl reductase from the methylotrophic yeast Ogataea minuta can catalyze the regio- and enantio-selective reduction of prochiral ketones to chiral alcohols, and is available for industrial manufacturing of statin drugs. We previously conducted a directed evolution experiment of the enzyme, and obtained a mutant (OCR_V166A) with improved tolerance to organic solvents. This expanded the applicability of the enzyme to the bioconversion of water-insoluble compounds (Honda etal., J. Biosci. Bioeng., 123, 673-678, 2017). In the present study, we expressed OCR_V166A in Rhodococcus opacus cells, which have a highly lipophilic surface structure and are dispersible in anhydrous organic solvents, and developed a whole-cell biocatalyst which can function in an organic-solvent-based reaction medium. The secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (TeADH) was employed as an NADPH-regenerating enzyme and co-expressed with OCR_V166A in R. opacus. The whole-cell bioconversion of 2,2,2-trifluoroacetophenone to α-(trifluoromethyl)benzyl alcohol was performed in organic solvents, including isopropanol, isobutanol, and cyclohexanol, which served both as reaction media and as substrates for TeADH. The type of organic solvents markedly affected not only the product titer but also the enantio-purity of the product. When isobutanol was used as the reaction medium, the whole-cell biocatalyst showed higher stability than the isolated enzyme. Consequently, a high concentration (1M) of the substrate was converted to the product with an overall conversion yield of 81% (mol/mol) in 24h.

Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously.

Cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates. Targeted integration of xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H2 and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo-oligomers (with degree of polymerization of 2-7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co-utilizing both cellulose and hemicellulose for the production of fuels and chemicals.

Biotechnological products in batch reactors obtained from cellulose, glucose and xylose using thermophilic anaerobic consortium

Abstract Lignocellulosic residues used for biofuel production is an alternative source to increase the energy supply. However, the cellulose found in this biomass must be made available in fermentable sugars, requiring a complex enzymatic mechanism only found in specific microorganisms. Some thermophilic and anaerobic bacteria of the Clostridium species are able to produce cellulolytic enzymes and metabolize pentose and hexose to ethanol. Therefore, we evaluated the degradation of cellulose, glucose, and xylose through the use of a thermophilic microbial consortium and ethanol production. The batch tests were performed in Thermoanaerobacter ethanolicus medium at 55 °C, pH 7. The tests were performed using 5.51 mmol/L glucose, 8.06 mmol/L xylose and 1 g/L cellulose. The highest ethanol yield was observed in the reactor with glucose (1.73 mol-EtOH/mol-glucose), followed by the reactor with xylose (1.33 mol-EtOH/mol-xylose). In contrast, the reactor with cellulose exhibited lower ethanol yield (1.88.10−3 mol-EtOH/g-cellulose), acetic acid and methane were also observed. Bacteria similar to Caloramator sp., Fervidobacterium sp., Thermoanaerobacterium sp. and Ethanoligenens sp. were identified by Illumina MiSeq sequencing, all related to the ethanol production.

Two‐Enzyme Hydrogen‐Borrowing Amination of Alcohols Enabled by a Cofactor‐Switched Alcohol Dehydrogenase

AbstractThe NADPH‐dependent secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (TeSADH), displaying broad substrate specificity and low enantioselectivity, was engineered to accept NADH as a cofactor. The engineered TeSADH showed a >10 000‐fold switch from NADPH towards NADH compared to the wildtype enzyme. This TeSADH variant was applied to a biocatalytic hydrogen‐borrowing system that employed catalytic amounts of NAD+, ammonia, and an amine dehydrogenase, which thereby enabled the conversion a range of alcohols into chiral amines.

Direct Alkylation of Amines with Primary and Secondary Alcohols through Biocatalytic Hydrogen Borrowing.

The reductive aminase from Aspergillus oryzae (AspRedAm) was combined with a single alcohol dehydrogenase (either metagenomic ADH-150, an ADH from Sphingobium yanoikuyae (SyADH), or a variant of the ADH from Thermoanaerobacter ethanolicus (TeSADH W110A)) in a redox-neutral cascade for the biocatalytic alkylation of amines using primary and secondary alcohols. Aliphatic and aromatic secondary amines were obtained in up to 99 % conversion, as well as chiral amines directly from the racemic alcohol precursors in up to >97 % ee, releasing water as the only byproduct.

Direct Alkylation of Amines with Primary and Secondary Alcohols through Biocatalytic Hydrogen Borrowing

AbstractThe reductive aminase from Aspergillus oryzae (AspRedAm) was combined with a single alcohol dehydrogenase (either metagenomic ADH‐150, an ADH from Sphingobium yanoikuyae (SyADH), or a variant of the ADH from Thermoanaerobacter ethanolicus (TeSADH W110A)) in a redox‐neutral cascade for the biocatalytic alkylation of amines using primary and secondary alcohols. Aliphatic and aromatic secondary amines were obtained in up to 99 % conversion, as well as chiral amines directly from the racemic alcohol precursors in up to >97 % ee, releasing water as the only byproduct.

Mutagenesis of Met-151 and Thr-153 to alanine in Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase changes substrate specificity for acetophenones.

Secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus reduces ketones to chiral alcohols, and generally obeys Prelog's Rule, with binding pockets for large and small alkyl substituents, giving (S)-alcohols. We have previously shown that mutations in both the large and small pockets can alter both substrate specificity and stereoselectivity. In the present work, Met-151 and Thr-153, residues located in the small pocket, were mutated to alanine. The M151A mutant SADH shows significantly lower activity and lower stereoselectivity for reduction of aliphatic ketones than wild-type SADH. Furthermore, M151A showed non-linear kinetics for reduction of acetone. T153A SADH shows lower activity but similar stereoselectivity for ketone reduction compared to wild-type SADH. The I86A/M151A/C295A and I86A/T153A/C295A triple mutant SADH show altered specificity for reduction of substituted acetophenones. These results confirm that these mutations are useful to combine with I86A/C295A SADH to expand the small pocket of SADH and broaden the substrate specificity.

Determining the roles of the three alcohol dehydrogenases (AdhA, AdhB and AdhE) in Thermoanaerobacter ethanolicus during ethanol formation.

Thermoanaerobacter ethanolicus is a promising candidate for biofuel production due to the broad range of substrates it can utilize and its high ethanol yield compared to other thermophilic bacteria, such as Clostridium thermocellum. Three alcohol dehydrogenases, AdhA, AdhB and AdhE, play key roles in ethanol formation. To study their physiological roles during ethanol formation, we deleted them separately and in combination. Previously, it has been thought that both AdhB and AdhE were bifunctional alcohol dehydrogenases. Here we show that AdhE has primarily acetyl-CoA reduction activity (ALDH) and almost no acetaldehyde reduction (ADH) activity, whereas AdhB has no ALDH activity and but high ADH activity. We found that AdhA and AdhB have similar patterns of activity. Interestingly, although deletion of both adhA and adhB reduced ethanol production, a single deletion of either one actually increased ethanol yields by 60-70%.

In vitro biocatalytic pathway design: orthogonal network for the quantitative and stereospecific amination of alcohols.

The direct and efficient conversion of alcohols into amines is a pivotal transformation in chemistry. Here, we present an artificial, oxidation-reduction, biocatalytic network that employs five enzymes (alcohol dehydrogenase, NADP-oxidase, catalase, amine dehydrogenase and formate dehydrogenase) in two concurrent and orthogonal cycles. The NADP-dependent oxidative cycle converts a diverse range of aromatic and aliphatic alcohol substrates to the carbonyl compound intermediates, whereas the NAD-dependent reductive aminating cycle generates the related amine products with >99% enantiomeric excess (R) and up to >99% conversion. The elevated conversions stem from the favorable thermodynamic equilibrium (K'eq = 1.88 × 1042 and 1.48 × 1041 for the amination of primary and secondary alcohols, respectively). This biocatalytic network possesses elevated atom efficiency, since the reaction buffer (ammonium formate) is both the aminating agent and the source of reducing equivalents. Additionally, only dioxygen is needed, whereas water and carbonate are the by-products. For the oxidative step, we have employed three variants of the NADP-dependent alcohol dehydrogenase from Thermoanaerobacter ethanolicus and we have elucidated the origin of the stereoselective properties of these variants with the aid of in silico computational models.