Recombinant Alcohol Dehydrogenase Research Articles

An Efficient Biocatalytic Preparation of a Key Chiral Intermediate to the Anti‐Epileptic Drug Cenobamate Using an In‐House Recombinant Alcohol Dehydrogenase

AbstractIn this investigation, the preparation of the chiral intermediate, (R)‐1‐(2‐chlorophenyl)‐2‐(2 H‐tetrazol‐2‐yl)ethan‐1‐ol (2 a), a precursor to the anti‐epileptic drug Cenobamate, was demonstrated using a very efficient alcohol dehydrogenase (ADH) enzyme from a simple ketone substrate. The enzyme, ADH107604 from Streptomyces coelicolor (ScADH), identified from Dr. Reddy's in‐house collection of biocatalysts, showed excellent activity against the ketone 1‐(2‐chlorophenyl)‐2‐(2 H‐tetrazol‐2‐yl)ethan‐1‐one 1, and the (R)‐alcohol product 2 a was obtained with excellent conversion and enantiomeric excess (ee) >99 %.

Hypoxia and lactate influence VOC production in A549 lung cancer cells.

Introduction: Cancer cells emit characteristic volatile organic compounds (VOCs), which are potentially generated from ROS-based lipid peroxidation of polyunsaturated fatty acids. The metabolism of such VOCs and their regulation remain to be fully investigated. In fact, the enzymes involved in the synthesis of these VOCs have not been described yet. Methods: In this study, we firstly conducted in vitro enzyme assays and demonstrated that recombinant alcohol dehydrogenase (ADH) converted Trans 2-hexenal into Trans 2-hexenol. The latter has previously been reported as a cancer VOC. To study VOC metabolism, 14 different culture conditions were compared in view of Trans 2-hexenol production. Results and discussion: The data indicate that hypoxia and the addition of lactate positively influenced Trans 2-hexenol production in A549 cancer cells. The RNAseq data suggested certain gene expressions in the VOC pathway and in lactate signaling, parallel to VOC production. This implies that hypoxia and lactate signaling with a VOC production can be characteristic for cancer in vitro.

Chemoenzymatic Synthesis of Tenofovir.

We report on novel chemoenzymatic routes toward tenofovir using low-cost starting materials and commercial or homemade enzyme preparations as biocatalysts. The biocatalytic key step was accomplished either via stereoselective reduction using an alcohol dehydrogenase or via kinetic resolution using a lipase. By employing a suspension of immobilized lipase from Burkholderia cepacia (Amano PS-IM) in a mixture of vinyl acetate and toluene, the desired (R)-ester (99% ee) was obtained on a 500 mg scale (60 mM) in 47% yield. Alternatively, stereoselective reduction of 1-(6-chloro-9H-purin-9-yl) propan-2-one (84 mg, 100 mM) catalyzed by lyophilized E. coli cells harboring recombinant alcohol dehydrogenase (ADH) from Lactobacillus kefir (E. coli/Lk-ADH Prince) allowed one to reach quantitative conversion, 86% yield and excellent optical purity (>99% ee) of the corresponding (R)-alcohol. The key (R)-intermediate was transformed into tenofovir through "one-pot" aminolysis-hydrolysis of (R)-acetate in NH3-saturated methanol, alkylation of the resulting (R)-alcohol with tosylated diethyl(hydroxymethyl) phosphonate, and bromotrimethylsilane (TMSBr)-mediated cleavage of the formed phosphonate ester into the free phosphonic acid. The elaborated enzymatic strategy could be applicable in the asymmetric synthesis of tenofovir prodrug derivatives, including 5'-disoproxil fumarate (TDF, Viread) and 5'-alafenamide (TAF, Vemlidy). The molecular basis of the stereoselectivity of the employed ADHs was revealed by molecular docking studies.

Enhancement of solubility of recombinant alcohol dehydrogenase from Rhodococcus ruber using predictive tool.

Solubility is one of key factors influencing the heterologous production of recombinant proteins in biotechnology. Among many aggregation-prone proteins, alcohol dehydrogenase (ADH-A) from Rhodococcus ruber (in this work abbreviated RrADH) shows a great potential in processes involved in the biotransformation of natural compounds. As ADH-A is a potentially high value asset in industrial biotransformation processes, improvement of its solubility would be of major commercial benefit. Predictive tools and in silico analysis provide a fast means for improving protein properties, for selecting appropriate changes, and ultimately for saving costs. We have therefore focused on enhancement of the solubility of RrADH using an online accesible predictive tool Aggrescan 3D 2.0. Selected mutations were introduced into the protein amino acid sequence by using site-directed PCR. This led to a 17% increase in the protein solubility of RrADHmut1 and a 98% increase for RrADHmut2. Moreover, the basic kinetics of the enzyme reaction were positively affected, further optimizing the overall performance of the production process.

Enhanced Acetate Tolerance and Recombinant Protein Accumulation in Escherichia coli by Transgenic Expression of a Heat Shock Protein from Carrot (Daucus carota L.).

In Escherichia coli (E. coli) culture, acetate accumulates as an undesirable by-product of aerobic fermentation on glucose and inhibits cell growth and recombinant protein production. We examined whether the heterologous expression of a eukaryotic heat shock protein (Hsp) can confer tolerance to acetate in E. coli. Transgenic cell lines (TCLs) heterologously expressing a small heat shock protein (sHsp) from carrot (Daucus carota L.), DcHsp17.7, were exposed to heat, sodium acetate, and alkaline conditions. The cell growth and cell viability were examined by measuring O.D.600 and colony-forming units (CFU), respectively. The His-tagged recombinant alcohol dehydrogenase (ADH) gene cloned in a pET11a expression vector was introduced into TCL1 and expressed by isopropyl β-D-1-thiogalactopyranoside treatment. After purifying using Ni-NTA affinity chromatography, its accumulation levels were examined using SDS-PAGE in the presence of acetate. TCLs constitutively expressing DcHsp17.7 showed improved growth, cell density, and cell viability under the stress conditions of heat, acetate, and alkaline compared to an empty vector control line. In acetate stress conditions, TCL1 accumulated more cellular proteins (approximately 130%) than the control. The recombinant ADH accumulated to a higher level in TCL1 (2.2-fold at 16 °C) than the control. The addition of acetate reduced the recombinant ADH level by 70% in the control when compared with the absence of acetate. In contrast, recombinant ADH accumulation was not affected by acetate in TCL1. In the presence of acetate, TCL1 accumulated 6.4-fold more recombinant ADH than did the control. Furthermore, recombinant ADH produced in TCL1 showed 1.5-fold higher enzyme activity than that produced in the control in the presence or absence of acetate. Our study showed that heterologously expressed DcHsp17.7 from carrot can alleviate the negative effects of acetate on E. coli.

Recombinant Enzymatic Redox Systems for Preparation of Aroma Compounds by Biotransformation.

The aim of this study was to develop immobilized enzyme systems that reduce carbonyl compounds to their corresponding alcohols. The demand for natural aromas and food additives has been constantly growing in recent years. However, it can no longer be met by extraction and isolation from natural materials. One way to increase the availability of natural aromas is to prepare them by the enzymatic transformation of suitable precursors. Recombinant enzymes are currently being used for this purpose. We investigated trans-2-hexenal bioreduction by recombinant Saccharomyces cerevisiae alcohol dehydrogenase (ScADH1) with simultaneous NADH regeneration by recombinant Candida boidinii formate dehydrogenase (FDH). In a laboratory bioreactor with two immobilized enzymes, 88% of the trans-2-hexenal was transformed to trans-2-hexenol. The initial substrate concentration was 3.7 mM. The aldehyde destabilized ScADH1 by eluting Zn2+ ions from the enzyme. A fed-batch operation was used and the trans-2-hexenal concentration was maintained at a low level to limit the negative effect of Zn2+ ion elution from the immobilized ScADH1. Another immobilized two-enzyme system was used to reduce acetophenone to (S)-1-phenylethanol. To this end, the recombinant alcohol dehydrogenase (RrADH) from Rhodococcus ruber was used. This biocatalytic system converted 61% of the acetophenone to (S)-1-phenylethanol. The initial substrate concentration was 8.3 mM. All enzymes were immobilized by poly-His tag to Ni2+, which formed strong but reversible bonds that enabled carrier reuse after the loss of enzyme activity.

Enhanced Production of Recombinant Alcohol Dehydrogenase Using the Genetically Engineered Escherichia coli Strain that Heterologously Expresses Carrot Heat Shock Protein 70.

Escherichia coli (E. coli) has been widely used as a host organism for producing recombinant proteins such as biocatalysts, antibody fragments, and therapeutic hormones. To enhance recombinant protein production, many E. coli strains have been genetically engineered on practical purposes. In this study, we developed the engineered E. coli strain expressing Heat shock protein 70, DcHsp70, from carrot (Daucus carota L.). The DNA construct for DcHsp70 expression, Lipoprotein promoter-DcHsp70 gene-Flippase recognition target cassette, which is flanked by the insertion site yddE pseudogene sequences, was generated by overlap PCR and inserted into the E. coli genome by lambda Red-mediated homologous recombination. To examine if the engineered E. coli cells can effectively produce recombinant proteins, the alcohol dehydrogenase (ADH) gene from a thermophile, Geobacillus stearothermophilus, was cloned into a pET11a expression vector and expressed by isopropyl β-D-1-thiogalactopyranoside treatment. Compared to wild type, the genetically engineered E. coli expressing DcHsp70 exhibited up to approximately 11-fold higher production of his-tagged ADH, mostly in soluble forms. The his-ADH protein that was purified from the engineered cells exhibited the enzyme activity. The genetically engineered E. coli developed in this study can be useful for the efficient production of recombinant proteins, such as recombinant ADH.

In silico analysis of suitable signal peptides for secretion of a recombinant alcohol dehydrogenase with a key role in atorvastatin enzymatic synthesis.

An elevated cholesterol level might lead to cardiovascular disease (CVD). Statins block the cholesterol synthesis pathway in the liver. Atorvastatin is the most widespread statin worldwide and, its chemical synthesis requires toxic catalysts, resulting in environmental pollution. Hence, enzymatic synthesis of atorvastatin is desirable. This process could be done by Lactobacillus kefir alcohol dehydrogenase (LKADH). Therefore, recombinant enzyme secretion by Escherichia coli using signal peptides (SPs) might result in easy production and purification. To achieve this objective, we used some online bioinformatics web servers to evaluate the suitable SPs for translocation of LKADH into extracellular spaces. “Signal Peptide Website” and “UniProt” were utilized to retrieve the SPs and LKADH sequences. “SignalP 4.1” was used to determine SPs and their cleavage site location and the results were rechecked by “Philius”. Physicochemical features of SPs were evaluated by “ProtParam”, then solubility of their fusion with LKADH was assessed by “Protein-sol”. Finally, secretion pathway and sub-cellular localization of the selected stable and soluble LKADH fusions were predicted by “PRED-TAT” and “ProtCompB”. Amongst the 41 evaluated SPs, only LPTA_ECOLI, SUBF_BACSU, CHIS_BACSU, SACB_BACAM, CDGT_BACST and AMY_BACLI could translocate LKADH out of cytoplasm. The six selected SPs in the result section were suitable to design a soluble secretory LKADH that accelerate its scale-up production and might be useful in future experimental researches.

The alcohol dehydrogenase with a broad range of substrate specificity regulates vitality and reproduction of the plant-parasitic nematode Bursaphelenchus xylophilus.

Pine wilt disease, which is caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, has caused huge damage to pine forests around the world. In this study, we analysed the PWN transcriptome to investigate the expression of genes related to the associated bacterial species Pseudomonas fluorescens and found that the gene adh-1 encoding alcohol dehydrogenase (ADH) was upregulated. The open reading frame of adh-1, which encoded a protein of 352 amino acid residues, was cloned from B. xylophilus. Recombinant ADH with a relative molecular weight of 39 kDa, was present mainly in inclusion bodies and was overexpressed in Escherichia coli BL21 (DE3) and purified after refolding. The biochemical assay revealed that recombinant ADH could catalyse the dehydrogen reaction of eight tested alcohols including ethanol in the presence of NAD+. Quantitative real-time RT-PCR analysis indicated that ethanol upregulated adh-1 expression in PWN. Results of RNA interference and inhibition of ADH treatment indicated that downregulating expression of adh-1 or inhibition of ADH could reduce ethanol tolerance and the vitality and reproduction ability of B. xylophilus, suggesting that adh-1 is involved in pathogenicity of PWN.

Arabidopsis thaliana alcohol dehydrogenase is differently affected by several redox modifications.

In plant cells, many stresses, including low oxygen availability, result in a higher production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). These molecules can lead to redox-dependent post-translational modification of proteins Cys residues. Here, we studied the effect of different redox modifications on alcohol dehydrogenase (ADH) from Arabidopsis thaliana. ADH catalyzes the last step of the ethanol fermentation pathway used by plants to cope with energy deficiency during hypoxic stress. Arabidopsis suspension cell cultures showed decreased ADH activity upon exposure to H2O2, but not to the thiol oxidizing agent diamide. We purified recombinant ADH and observed a significant decrease in the enzyme activity by treatments with H2O2 and diethylamine NONOate (DEA/NO). Treatments leading to the formation of a disulfide bond between ADH and glutathione (protein S-glutathionylation) had no negative effect on the enzyme activity. LC-MS/MS analysis showed that Cys47 and Cys243 could make a stable disulfide bond with glutathione, suggesting redox sensitivity of these residues. Mutation of ADH Cys47 to Ser caused an almost complete loss of the enzyme activity while the Cys243 to Ser mutant had increased specific activity. Incubation of ADH with NAD+ or NADH prevented inhibition of the enzyme by H2O2 or DEA/NO. These results suggest that binding of ADH with its cofactors may limit availability of Cys residues to redox modifications. Our study demonstrates that ADH from A. thaliana is subject to different redox modifications. Implications of ADH sensitivity to ROS and RNS during hypoxic stress conditions are discussed.

Expression of recombinant alcohol dehydrogenase with attached glycans in Escherichia coli

Expression of recombinant alcohol dehydrogenase with attached glycans in Escherichia coli

Cloning and characterization of thermostable GroEL/GroES homologues from Geobacillus thermopakistaniensis and their applications in protein folding

The chaperonin genes encoding GroELGt (ESU72018) and GroESGt (ESU72017), homologues of bacterial GroEL and GroES, from Geobacillus thermopakistaniensis were cloned and expressed in Escherichia coli. The purified gene products possessed the ATPase activity similar to other bacterial and eukaryal counterparts. Recombinant GroELGt and GroESGt were able to refold the denatured insoluble aggregates of α-amylase from Bacillus licheniformis into soluble and active form. Furthermore, GroELGt and GroESGt successfully enhanced the thermostability of porcine heart malate dehydrogenase. Expression of GroELGt gene in E. coli cells enhanced the thermotolerance of the host. Furthermore, soluble production of recombinant alcohol dehydrogenase from Bacillus subtilis strain R5 in E. coli, initially produced as insoluble aggregates, was achieved by co-expressing the gene with GroELGt. Our results implied that GroELGt could assist folding of nascent protein in E. coli with the help of host co-chaperonin without requiring additional ATP. This system can be used for soluble production of recombinant proteins which otherwise are produced in insoluble form in E. coli. To the best of our knowledge this is the first report on functional characterization and applications of chaperonins from genus Geobacillus.

Synthesis of 1-(S)-phenylethanol and ethyl (R)-4-chloro-3-hydroxybutanoate using recombinant Rhodococcus erythropolis alcohol dehydrogenase produced by two yeast species

The ReADH gene of Rhodococcus erythropolis DSM 43297 encoding alcohol dehydrogenase (ReADH) was expressed in the yeast Arxula adeninivorans and Hansenula polymorpha to determine which host accumulated the highest concentration of recombinant alcohol dehydrogenase for the production of enantiometrically pure alcohols. The ReADH gene was fused with a His-tag encoding sequence at its 3′-end and expressed in both yeast species. The recombinant ReADH-6H preparations exhibited small host-strain dependent differences in their pH and temperature optima, thermo-stability and substrate specificity. The recombinant enzymes in combination with a substrate-coupled cofactor regeneration system were used to synthesize 1-(S)-phenylethanol and ethyl (R)-4-chloro-3-hydroxybutanoate. Permeabilized A. adeninivorans whole cell catalysts co-expressing R. erythropolis alcohol dehydrogenase and Bacillus megaterium glucose dehydrogenase, for enzyme-coupled cofactor regeneration, were also used for the synthesis reactions.

Characterization of alcohol dehydrogenase from Kangiella koreensis and its application to production of all-trans-retinol.

A recombinant alcohol dehydrogenase (ADH) from Kangiella koreensis was purified as a 40 kDa dimer with a specific activity of 21.3 nmol min(-1) mg(-1), a K m of 1.8 μM, and a k cat of 1.7 min(-1) for all-trans-retinal using NADH as cofactor. The enzyme showed activity for all-trans-retinol using NAD (+) as a cofactor. The reaction conditions for all-trans-retinol production were optimal at pH 6.5 and 60 °C, 2 g enzyme l(-1), and 2,200 mg all-trans-retinal l(-1) in the presence of 5% (v/v) methanol, 1% (w/v) hydroquinone, and 10 mM NADH. Under optimized conditions, the ADH produced 600 mg all-trans-retinol l(-1) after 3 h, with a conversion yield of 27.3% (w/w) and a productivity of 200 mg l(-1) h(-1). This is the first report of the characterization of a bacterial ADH for all-trans-retinal and the biotechnological production of all-trans-retinol using ADH.

Production of a thermostable alcohol dehydrogenase from Rhodococcus ruber in three different yeast species using the Xplor®2 transformation/expression platform

The Xplor®2 transformation/expression platform was employed for comparative assessment of three different yeast species as hosts for synthesis of a thermostable nicotinamide adenine dinucleotide (NAD⁺)-dependent medium-chain alcohol dehydrogenase from Rhodococcus ruber strain 219. Using yeast ribosomal DNA (rDNA) integrative expression cassettes (YRCs) and yeast integrative expression cassettes (YICs) equipped with a selection-marker module and one, two or four expression modules for transformation of auxotrophic Arxula adeninivorans, Hansenula polymorpha, and Saccharomyces cerevisiae strains, quantitative comparison of the yield of recombinant alcohol dehydrogenase RR-ADH6Hp in all three species was carried out. In all cases, the RR-ADH6H gene was expressed under the control of the strong constitutive A. adeninivorans-derived TEF1 promoter, which functions in all yeast species analyzed. Recombinant RR-ADH6Hp accumulated intracellularly in all strains tested. The best yields of active enzyme were obtained from A. adeninivorans, with S. cerevisiae producing intermediate amounts. Although H. polymorpha was the least efficient producer overall, the product obtained was most similar to the enzyme synthesized by R. ruber 219 with respect to its thermostability.

Glucose-limited high cell density cultivations from small to pilot plant scale using an enzyme-controlled glucose delivery system

The enzyme controlled substrate delivery cultivation technology EnBase(®) Flo allows a fed-batch-like growth in batch cultures. It has been previously shown that this technology can be applied in small cultivation vessels such as micro- and deep well plates and also shake flasks. In these scales high cell densities and improved protein production for Escherichia coli cultures were demonstrated. This current study aims to evaluate the scalability of the controlled glucose release technique to pilot scale bioreactors. Throughout all scales, that is, deep well plates, 3 L bioreactor and 150 L bioreactor cultivations, the growth was very similar and the model protein, a recombinant alcohol dehydrogenase (ADH) was produced with a high yield in soluble form. Moreover, EnBase Flo also was successfully used as a controlled starter culture in high cell density fed-batch cultivations with external glucose feeding. Here the external feeding pump was started after overnight cultivation with EnBase Flo. Final optical densities in these cultivations reached 120 (corresponding to about 40 g L(-1) dry cell weight) and a high expression level of ADH was obtained. The EnBase cultivation technology ensures a controlled initial cultivation under fed-batch mode without the need for a feeding pump. Because of the linear cell growth under glucose limitation it provides optimal and robust starting conditions for traditional external feed-based processes.

Enhanced growth and recombinant protein production of Escherichia coli by a perfluorinated oxygen carrier in miniaturized fed-batch cultures

BackgroundLiquid perfluorochemicals (PFCs) are interesting oxygen carriers in medicine and biotechnology with a high solubility for oxygen. They have been repeatedly used for improving oxygen transfer into prokaryotic and eukaryotic cell cultures, however their application is still limited. Here we show the great benefit of air/oxygen saturated perfluorodecalin (PFD) for high cell density cultivation of Escherichia coli in microwell plates and their positive effect on the soluble production of a correctly folded heterologously expressed alcohol dehydrogenase.ResultsIn EnBase® cultivations the best effect was seen with PFD saturated with oxygen enriched air (appr. 10 μM oxygen per ml) when PFD was added at the time of induction. In contrast the effect of PFD was negligible when it was added already at the time of inoculation. Optimisation of addition time and content of loaded oxygen into the PFD resulted in an increased the cell density by 40% compared to control cultures, and correspondingly also the product yield increased, demonstrated at the example of a recombinant alcohol dehydrogenase.ConclusionsPFCs are a valuable additive in miniaturized cell culture formats. For production of recombinant proteins in low cell density shaken cultures the addition of oxygen-enriched PFD makes the process more robust, i.e. a high product yield is not any more limited to a very narrow cell density window during which the induction has to be done. The positive effect of PFD was even more obvious when it was added during high cell density cultures. The effect of the PFD phase depends on the amount of oxygen which is loaded into the PFD and which thus is a matter of optimisation.

Erratum to: Purification and biochemical characterization of recombinant alcohol dehydrogenase from the psychrophilic bacterium Pseudomonas frederiksbergensis

A gram-negative psychrophilic bacterium, with potential for biodegradation of long-chain n-alkanes was isolated from ice samples collected in Spitzbergen, Denmark. On the basis of results of biochemical and morphological tests and sequence analysis of 16S rRNA, the strain was identified as Pseudomonas frederiksbergensis. In this work, a short-chain NAD+-dependent alcohol dehydrogenase (alcDH) (Accession number: AAR13804) from the P. frederiksbergensis was cloned and transformed in E. coli BL21 (3DE) competent cells. The alcDH activity was highest in the crude extract of cells induced with 1.0 mM IPTG. The recombinant alcDH enzyme was purified to 93.4% homogeneity using three consecutive purification steps including ammonium sulphate, Q-Sepharose Fast Flow column and gel filtration chromatography employing Superdex 200 10/30 HR column. Enzyme enrichment and yield levels of 31.4 folds and 25.5%, respectively, were achieved. While the subunit molecular mass of the enzyme was determined on SDS-PAGE to be ~38 kDa, the aggregated native form of the enzyme had a molecular mass of ~238 kDa by gel filtration analysis. Reaction conditions optima for the recombinant alcDH were determined with propan-1-ol as the substrate. While the optimum pH was 9, the optimum temperature was 35 °C. The alcDH enzyme exhibited moderate thermal stability with half-lives of 150 min at 55 °C, 27 min at 65 °C and 8 min at 75 °C. Results for kinetic parameters indicated that the apparent K m value for alcDH with propan-1-ol as the substrate was found to be 1.42 mM and the V max value was 0.63 mmol mg−1 min−1. Experimental evidence revealed that the recombinant alcDH exhibited a wide range of substrate specificity, with higher levels of specific activity for aliphatic alcohols as compared to secondary alcohols. Taken together, the present study highlights the potential of alcDH as a member of cold-adapted enzymes in several key biotechnological applications including environmental bioremediation and biotransformations. It is envisaged that, with the ongoing screening of microorganisms and metagenomes, directed evolution approaches and the subsequent overexpression of recombinant proteins, more enzymes will be found that are suitable for bioremediation purposes.

High-yield production of biologically active recombinant protein in shake flask culture by combination of enzyme-based glucose delivery and increased oxygen transfer

This report describes the combined use of an enzyme-based glucose release system (EnBase®) and high-aeration shake flask (Ultra Yield Flask™). The benefit of this combination is demonstrated by over 100-fold improvement in the active yield of recombinant alcohol dehydrogenase expressed in E. coli. Compared to Terrific Broth and ZYM-5052 autoinduction medium, the EnBase system improved yield mainly through increased productivity per cell. Four-fold increase in oxygen transfer by the Ultra Yield Flask contributed to higher cell density with EnBase but not with the other tested media, and consequently the product yield per ml of EnBase culture was further improved.

Purification and biochemical characterization of recombinant alcohol dehydrogenase from the psychrophilic bacterium Pseudomonas frederiksbergensis

Purification and biochemical characterization of recombinant alcohol dehydrogenase from the psychrophilic bacterium Pseudomonas frederiksbergensis