Cell Biocatalyst Research Articles

Autodisplay of glucose-6-phosphate dehydrogenase for redox cofactor regeneration at the cell surface.

Inherent cofactor regeneration is a pivotal feature of whole cell biocatalysis. For specific biotechnological applications, surface display of enzymes is emerging as a tool to circumvent mass transfer limitations or enzyme stability problems. Even complex reactions can be accomplished applying displayed enzymes. Yet, industrial utilization of the technique is still impeded by lacking cofactor regeneration at the cell surface. Here, we report on the surface display of a glucose-6-phoshate dehydrogenase (G6PDH) via Autodisplay to address this limitation and regenerate NADPH directly at the cell surface. The obtained whole cell biocatalyst demonstrated similar kinetic parameters compared to the purified enzyme, more precisely KM values of 0.2 mM for NADP+ and calculated total turnover numbers of 107 . However, the KM for the substrate G6P increased by a factor of 7 to yield 1.5 mM. The whole cell biocatalyst was cheaper to produce, easy to separate from the reaction mixture and reusable in consecutive reaction cycles. Furthermore, lyophilization allowed storage at room temperature. The whole cell biocatalyst displaying G6PDH was applicable for NADPH regeneration in combination with soluble as well as surface displayed enzymes and model reactions in combination with bacterial CYP102A1 and human CYP1A2 were realized. Biotechnol. Bioeng. 2017;114: 1658-1669. © 2017 Wiley Periodicals, Inc.

Reductive amination of ketones catalyzed by whole cell biocatalysts containing imine reductases (IREDs)

The asymmetric reductive amination of ketones represents an elegant and convenient way to obtain chiral amines. Recently, several examples have been reported in which isolated imine reductases (IREDs) have been used for this type of reaction leading to promising results. In this work we focus on the applicability of whole cell biocatalysts (recombinant E. coli cells heterologously overexpressing an IRED) to simplify its preparation and to cut on catalyst production costs. Thirteen IREDs were screened towards six different ketones, using methylamine as amine donor. The targeted amines were formed with low to very high conversions and good to excellent stereoselectivity, depending on both, the ketone amine pair used for the reaction, as well as the applied IRED. It was further proven that a micro-aqueous reaction environment was applicable showing similar activity trends for the various reductive aminations but predominantly reduced conversions. A preparative scale experiment in a buffered environment was conducted leading to 93% conversion and 99% stereoselectivity of the product (1S,3R)-N,3-dimethylcyclohexylamine. As the whole cells intrinsic glucose dehydrogenase could be used for cofactor regeneration, no enzyme addition had to be applied, making this biocatalyst formulation particularly cost efficient.

Continuous supply of glucose and glycerol enhances biotransformation of ricinoleic acid to (E)-11-(heptanoyloxy) undec-9-enoic acid in recombinant Escherichia coli

This study aimed at the development of biotransformation strategies with feeding of energy sources for bioconversion of ricinoleic acid to (E)-11-(heptanoyloxy) undec-9-enoic acid (11-HOUA), a key intermediate of brassylic acid, by recombinant Escherichia coli overexpressing an alcohol dehydrogenase from Micrococcus luteus and a Baeyer-Villiger monooxygenase from Pseudomonas putida KT2440. Feeding of glucose or glycerol facilitated both the preparation of high-density cell biocatalyst and supply of the NAD+ and NADPH cofactors. By the glucose feeding strategy, 30.8g/L of the engineered E. coli cells produced 29.7mM of 11-HOUA with 1.9mM/h of productivity, which were 1.8 and 1.6 times higher than the same biotransformation without the glucose feeding, respectively. Intermittent addition of glycerol increased 11-HOUA productivity by 16% compared to that by the glucose feeding. Finally, 34.5mM of 11-HOUA concentration, 77% conversion and 2.2mM/h productivity were obtained using 31.6g/L of cell biocatalyst along with the glycerol addition. It was concluded that supplementation of additional carbon sources in biotransformation process would be a potent strategy to increase the performance of fatty acid conversion.

Fungal biocatalyst activated by an electric field: Improved mass transfer and non-specificity for hydrocarbon degradation in an airlift bioreactor

The combination of biological and electrochemical techniques enhances the bioremediation efficiency of treating oil-contaminated water. In this study a non-growing fungal whole cell biocatalyst (BC; Aspergillus brasiliensis attached to perlite) pretreated with an electric field (EF), was used to degrade a hydrocarbon blend (hexadecane-phenanthrene-pyrene; 100:1:1w/w) in an airlift bioreactor (ALB). During hydrocarbon degradation, all mass transfer resistances (internal and external) and sorption capacity were experimentally quantified. Internal mass transfer resistances were evaluated through BC effectiveness factor analysis as a function of the Thiele modulus (using first order reaction kinetics, assuming a spherical BC, five particle diameters). External (interfacial) mass transfer resistances were evaluated by kLa determination. EF pretreatment during BC production promoted surface changes in BC and production of an emulsifier protein in the ALB. The BC surface modifications enhanced the affinity for hydrocarbons, improving hydrocarbon uptake by direct contact. The resulting emulsion was associated with decreased internal and external mass transfer resistances. EF pretreatment effects can be summarized as: a combined uptake mechanism (direct contact dominant followed by emulsified form dominant) diminishing mass transfer limitations, resulting in a non-specific hydrocarbon degradation in blend. The pretreated BC is a good applicant for oil-contaminated water remediation.

Phenylalanine ammonia lyase from Arabidopsis thaliana (AtPAL2): A potent MIO-enzyme for the synthesis of non-canonical aromatic alpha-amino acids.: Part II: Application in different reactor concepts for the production of (S)-2-chloro-phenylalanine

Phenylalanine ammonia lyase (PAL) from Arabidopsis thaliana (AtPAL2) is in general a very good catalyst for the amination of fluoro- and chloro-cinnamic acid derivatives yielding halogenated (S)-phenylalanine derivatives with ≥85% conversion and excellent ee values >99%. We have studied the application of this enzyme as whole cell biocatalyst and immobilized on the cellulose carrier Avicel® for the production of the hypertension drug precursor (S)-2-chloro-phenylalanine using batch, fed-batch, as well as continuous membrane reactor and plug-flow reactor. For immobilization, a C-terminal fusion of the enzyme with a carbohydrate binding module (CBM) was produced, which selectively binds to Avicel® directly from crude cell extracts, thus enabling a fast and cheap immobilization, stabilization and recycling of the enzyme. 1g Avicel was loaded with 10mg enzyme.Best results were obtained with whole cells using the continuous membrane reactor (47gproduct/gDryCellWeight) and using the immobilized enzyme in a repetitive fed-batch (274gproduct/gimmobilized enzyme) or in a continuous plug-flow reactor (288gproduct/gimmobilize enzyme). Therewith the productivity of AtPAL2 outperforms the established fed-batch process at DSM using PAL from Rhodotorula glutinis in E. coli as whole cell biocatalyst with a productivity of 0.14gproduct/gWetCellWeight (ca. 0.7gproduct/gDryCellWeight) (de Lange et al., 2011; doi:10.1002/cctc.201000435).

Biocatalytic activity of Monascus mycelia depending on physiology and high sensitivity to product concentration

Cell suspension culture using mycelia as whole cell biocatalyst for production of orange Monascus pigments has been carried out successfully in a nonionic surfactant micelle aqueous solution. Thus, selection of mycelia as whole cell biocatalyst and the corresponding enzymatic kinetics for production of orange Monascus pigments can be optimized independently. Mycelia selected from submerged culture in a nonionic surfactant micelle aqueous solution with low pH 2.5 exhibits robust bioactivity. At the same time, enzymatic kinetic study shows that the bioactivity of mycelia as whole cell biocatalyst is sensitive to high product concentration. Segregation of product from mycelia by cell suspension culture in a nonionic surfactant micelle aqueous solution or peanut oil–water two-phase system is not only necessary for studying the enzymatic kinetics but also beneficial to industrial application of mycelia as whole cell biocatalyst.

Optimized whole cell biocatalyst from acetoin to 2,3‐butanediol through coexpression of acetoin reductase with NADH regeneration systems in engineered Bacillus subtilis

AbstractBACKGROUND2,3‐Butanediol (2,3‐BD) has a wide range of applications in chiral molecular synthesis, biofuel additives, and in food flavor additive manufacturing. Fermentation is a favorable method for 2,3‐BD production. However, it requires much time and produces several NADH related byproducts which compete with 2,3‐BD production. Bacillus subtilis has an excellent ability for 2,3‐BD production by biocatalysis. However, its production is limited by low intracellular NADH and the reversible property of acetoin reductase (AR/2,3‐BDH). The whole cell biocatalyst process with two different NADH regeneration systems was designed for efficient production of 2,3‐BD in B. subtilis 168.RESULTSFormate dehydrogenase and glucose dehydrogenase for NADH regeneration were successfully co‐expressed with acetoin reductase in B. subtilis 168. After optimization of biocatalyst bioconversion conditions, B. subtilis 168/pMA5‐bdhA‐HpaII‐fdh yielded 74.5 g L−1 of 2, 3‐BD with 9.3 g L−1 h−1 productivity by fed batch and 115.4 g of 2,3‐BD was achieved using same batch bacterium by three repeated batch bioconversions. On the other hand, 63.7 g L−1 of 2, 3‐BD was produced with 7.92 g L−1 h−1 productivity by B. subtilis 168/pMA5‐bdhA‐HpaII‐gdh. To our knowledge, the volume productivity obtained here is the highest ever reported for biocatalysis.CONCLUSIONA higher productivity of 2,3‐BD from acetoin was achieved by whole cell biocatalysis with NADH regeneration systems in B. subtilis 168. This approach can be applied for NADH related bio‐based chemicals production to improve titer, yield and productivity. © 2017 Society of Chemical Industry

Phenylalanine ammonia lyase from Arabidopsis thaliana (AtPAL2): A potent MIO-enzyme for the synthesis of non-canonical aromatic alpha-amino acids: Part I: Comparative characterization to the enzymes from Petroselinum crispum (PcPAL1) and Rhodosporidium toruloides (RtPAL)

Phenylalanine ammonia lyase (PAL) from Arabidopsis thaliana (AtPAL2) was comparatively characterized to the well-studied enzyme from parsley (PcPAL1) and Rhodosporidium toruloides (RtPAL) with respect to kinetic parameters for the deamination and the amination reaction, pH- and temperature optima and the substrate range of the amination reaction. Whereas both plant enzymes are specific for phenylalanine, the bifunctional enzyme from Rhodosporidium toruloides shows KM-values for L-Phe and L-Tyr in the same order of magnitude and, compared to both plant enzymes, a 10–15-fold higher activity.At 30°C all enzymes were sufficiently stable with half-lives of 3.4days (PcPAL1), 4.6days (AtPAL2) and 9.7days (RtPAL/TAL). Very good results for the amination of various trans-cinnamic acid derivatives were obtained using E. coli cells as whole cell biocatalysts in ammonium carbonate buffer. Investigation of the substrate ranges gave interesting results for the newly tested enzymes from A. thaliana and R. toruloides. Only the latter accepts besides 4-hydroxy-CA also 3-methoxy-4-hydroxy-CA as a substrate, which is an interesting intermediate for the formation of pharmaceutically relevant L-Dopa.AtPAL2 is a very good catalyst for the formation of (S)-3-F-Phe, (S)-4-F-Phe and (S)-2-Cl-Phe. Such non-canonical amino acids are valuable building blocks for the formation of various drug molecules.

Enzymatic preparation of optically pure t -butyl 6-chloro-(3 R ,5 S )-dihydroxyhexanoate by a novel alcohol dehydrogenase discovered from Klebsiella oxytoca

Abstract Alcohol dehydrogenases can catalyze the inter-conversion of aldehydes and alcohols. The t -butyl 6-chloro-(3 R ,5 S )-dihydroxyhexanoate is a key chiral intermediate in the synthesis of statin-type drugs such as Crestor (rosuvastatin calcium) and Lipitor (atorvastatin). Herein, a novel alcohol dehydrogenase (named as KleADH) discovered from Klebsiella oxytoca by a genome mining method was cloned and characterized. The KleADH was functionally overexpressed in Escherichia coli Rosetta (DE3) and the whole cell biocatalyst was able to convert t -butyl 6-chloro-(5 S )-hydroxy-3-oxohexanoate to t -butyl 6-chloro-(3 R ,5 S )-dihydroxyhexanoate with more than 99% diastereomeric excess (de) and 99% conversion in 24 h without adding any expensive cofactors. Several factors influencing the whole cell catalyst activity such as temperature, pH, the effects of metal ions and organic solvent were determined. The optimum enzyme activity was achieved at 30 °C and pH 7.0 and it was shown that 1 mM Fe 3+ can increase the enzyme activity by 1.2 times. N-hexane/water and n-heptane/water biphasic systems can also increase the activity of KleADH. Substrate specificity studies showed that KleADH also exhibited notable activity towards several aryl ketones with high stereoselectivity. Our investigation on this novel alcohol dehydrogenase KleADH reveals a promising biocatalyst for producing chiral alcohols for preparation of valuable pharmaceuticals.

Influence of femtosecond laser produced nanostructures on biofilm growth on steel

Biofilm formation poses high risks in multiple industrial and medical settings. However, the robust nature of biofilms makes them also attractive for industrial applications where cell biocatalysts are increasingly in use. Since tailoring material properties that affect bacterial growth or its inhibition is gaining attention, here we focus on the effects of femtosecond laser produced nanostructures on bacterial adhesion. Large area periodic surface structures were generated on steel surfaces using 30-fs laser pulses at 790nm wavelength. Two types of steel exhibiting a different corrosion resistance were used, i.e., a plain structural steel (corrodible) and a stainless steel (resistant to corrosion). Homogeneous fields of laser-induced periodic surface structures (LIPSS) were realized utilizing laser fluences close to the ablation threshold while scanning the sample under the focused laser beam in a multi-pulse regime. The nanostructures were characterized with optical and scanning electron microscopy. For each type of steel, more than ten identical samples were laser-processed. Subsequently, the samples were subjected to microbial adhesion tests. Bacteria of different shape and adhesion behavior (Escherichia coli and Staphylococcus aureus) were exposed to laser structures and to polished reference surfaces. Our results indicate that E. coli preferentially avoids adhesion to the LIPSS-covered areas, whereas S. aureus favors these areas for colonization.

Unveiling the Biocatalytic Aromatizing Activity of Monoamine Oxidases MAO-N and 6-HDNO: Development of Chemoenzymatic Cascades for the Synthesis of Pyrroles

A chemoenzymatic cascade process for the sustainable production of pyrroles has been developed. Pyrroles were synthesized by exploiting the previously unexplored aromatizing activity of monoamine oxidase enzymes (MAO-N and 6-HDNO). MAO-N/6-HDNO whole cell biocatalysts are able to convert 3-pyrrolines into pyrroles under mild conditions and in high yields. Moreover, MAO-N can work in combination with the ruthenium Grubbs catalyst, leading to the synthesis of pyrroles from diallylamines/-anilines in a one-pot cascade metathesis–aromatization sequence.

Steroid Bioconversions.

Steroid modifications by selected wild-type and engineered strains of microorganisms became an effective tool for the production of high-valued steroidal drugs and their precursors for the pharmaceutical industry. Some microorganisms are effective at the performance of sterol side-chain degradation, oxyfunctionalization of steroid core, and redox reactions at different positions of the steroid molecule. A number of bioprocesses using steroid-transforming microbial strains are well established on an industrial level. Although a range of biocatalytic methods has been developed, selection of suitable microorganisms, as well as creation of new engineered strains, is of great importance for generation of improved bioprocesses and production schemes for obtaining known and new metabolites with potent biological activity. The achievements in genetic and metabolic engineering of steroid-transforming strains in combination with novel approaches in the enzymatic and whole-cell biocatalysis provide a platform for highly effective and selective biotransformations.Here, we briefly review the current state and prospects in the field of microbial bioconversions with special attention to the application of molecular microbiology methods for the generation of new whole cell biocatalysts.

Microfluidic Devices for Biotechnological Applications

Single cell analytics and new separation techniques are two examples of the fast growing field of microfluidics. Here, we present two microfluidic devices, containing 3D-confinements, for biotechnological or medical applications. The first device provides new purification methods of minicircle DNA (gene vaccines). We developed a dielectrophoresis based continuous-flow separation of DNA depending on the conformation, i.e. linear or supercoiled (ccc) form. Our second device is used for whole cell biocatalyst studies. We developed a microfluidic device for trapping and cultivation of E. coli. Additionally, we proved that UV spectroscopy can be exploited for label-free detection of cofactors, providing monitoring of cofactor leaching.

Unveiling the Hidden Performance of Whole Cells in the Asymmetric Bioreduction of Aryl‐containing Ketones in Aqueous Deep Eutectic Solvents

AbstractIn this contribution, we report the first successful baker's yeast reduction of arylpropanones using deep eutectic solvents (DESs) as biodegradable and non‐hazardous co‐solvents. The nature of DES [e.g. choline chloride/glycerol (2:1)] and the percentage of water in the mixture proved to be critical for both the reversal of selectivity and to achieve high enantioselectivity on going from pure water (up to 98:2 er in favour of theS‐enantiomer) to DES/aqueous mixtures (up to 98:2 er in favour of theR‐enantiomer). As a result, both enantiomers of valuable chiral alcohols of pharmaceutical interest were prepared from the same biocatalyst by simply switching the solvent. The possible inhibition of some (S)‐oxidoreductases making part of the genome of such a wild‐type whole cell biocatalyst when DESs are used as co‐solvents may pave the way for ananti‐Prelog reduction. The scope and limitations of this kind of biotransformations for a range of aryl‐containing ketones are also discussed.magnified image

Chemo-biocatalytic one-pot two-step conversion of cyclic amine to lactam using whole cell monoamine oxidase

BACKGROUND Most biocatalysts currently involved in one-pot chemoenzymatic cascades are pure enzymes, while whole cells and crude enzyme extracts remain unexplored. This work aims to develop a chemo-biocatalytic one-pot two-step system involving whole cell monoamine oxidase (MAO, EC 1.4.3.4) coupled with a Cu-based oxidative system (CuI/H2O2) for the transformation of 1,2,3,4-tetrahydroisoquinoline (THIQ) to 3,4-dihydroisoquinolin-1(2H)-one (DHIO). RESULTS MAO-N variants D9 and D11 were tested as whole cell and crude lysate biocatalysts for biological oxidation. Whole Escherichia coli OverExpress C43(DE3) cells expressing MAO-N D9 showed the best performance (Vmax = 36.58 mmol L−1 h−1, KM = 8.124 mmol L−1, maximum specific productivity 89.3 μmol min−1 g−1DCW) and were employed in combination with CuI/H2O2 in a sequential one-pot two-step process. The biotransformation was scaled-up to the initial volume of 25 mL and after triple THIQ feeding, 48.2 mmol L−1 of the intermediate 3,4-dihydroisoquinoline (DHIQ) was obtained with a yield of 71.3%. Afterwards, chemical catalysts (1 mol% CuI and 10 eq. H2O2) were added to the biologically produced DHIQ, which was transformed to ∼30 mmol L−1 DHIO at 69.4% overall yield. CONCLUSION As MAO-N variants have wide substrate specificity, this work broadens the portfolio of one-pot chemoenzymatic processes employing whole cell biocatalysts, representing an alternative to using pure enzymes. © 2016 Society of Chemical Industry

A whole cell biocatalyst for double oxidation of cyclooctane.

A novel whole cell cascade for double oxidation of cyclooctane to cyclooctanone was developed. The one-pot oxidation cascade requires only a minimum of reaction components: resting E. coli cells in aqueous buffered medium (=catalyst), the target substrate and oxygen as environmental friendly oxidant. Conversion of cyclooctane was catalysed with high efficiency (50% yield) and excellent selectivity (>94%) to cyclooctanone. The reported oxidation cascade represents a novel whole cell system for double oxidation of non-activated alkanes including an integrated cofactor regeneration. Notably, two alcohol dehydrogenases from Lactobacillus brevis and from Rhodococcus erythropolis with opposite cofactor selectivities and one monooxygenase P450 BM3 were produced in a coexpression system in one single host. The system represents the most efficient route with a TTN of up to 24363 being a promising process in terms of sustainability as well.

<b>Permeabilization of Saccharomyces fragilis IZ 275 cells with ethanol to obtain a biocatalyst with lactose hydrolysis capacity

The permeabilization was used to transform microorganisms in cell biocatalysts with high enzymatic activity. The Saccharomyces fragilis IZ 275 yeast cells were permeabilized with ethanol, as permeabilizing agent. To optimize the permeabilization conditions were used the design of Box-Behnken 15 trials (3 central points). The independent variables and their levels were ethanol (29, 32 and 35%), temperature (15, 20 and 25°C) and time (15, 20 and 25 min). The answer (Y) function has beta-galactosidase activity (U mg -1 ). The optimum conditions for obtaining a high enzymatic activity were observed in 35% ethanol concentration, temperature 15oC and 20 min. treatment time. The maximum activity of the enzyme beta-galactosidase obtained was 10.59 U mg -1 . The permeabilization of the S. fragilis IZ 275 cells was efficient.

Whole Cell Biocatalyst for Soyasapogenol B Production from Soybean Saponin

Soyasapogenol B (SB), aglycone of soybean saponin, is known to have hepatoprotective, antimutagenic, antivirus, and anti-inflammatory activities. This research examined the use of whole-cell biocatalyst to produce SB from soybean saponin. It was found that Aspergillus flavus, a fungus isolated from peanut pods, was capable of expressing extracellular and intracellular saponin hydrolase enzyme. However, the total enzyme activity produced using fungal whole cells (37U) in the reaction mixture was about 3 times that produced using the extracellular (12.4U) or intracellular (11.5U) enzyme. Cells with maximum hydrolytic activity for production of SB (12.2 U/g) was obtained using production medium supplemented by 2% soybean saponin, as inducer for enzyme production, adjusted at pH 9 and incubated at 30°C for 2 days. The highest yield of SB was achieved when the reaction mixture was incubated at pH 5.5 and 45°C for 48h; using 20% wet cells (corresponding to 4% cell dry weight) and soybean saponin (2%, w/v) as a substrate. Under these optimal conditions, the cells bioconversion efficiency (SB yield) increased from 5.3 to 60%. Whole cell biocatalyst has several advantages with regard to industrial applications: a consistent quality, easy to be prepared and a very low price compared with purified enzyme. Consequently, this study is significant for production of SB from soybean saponin on an industrial scale.

Effect of culture conditions on the whole cell activity of recombinant Escherichia coli expressing periplasmic organophosphorus hydrolase and cytosolic GroEL/ES chaperone

Specific whole cell activity strongly affects sensitivity and detection limit of whole cell-based biosensors. Previously, we developed recombinant Escherichia coli coexpressing periplasmic organophosphorus hydrolase (OPH) and cytosolic chaperone GroEL-GroES (GroEL/ES). In present work, we investigated the effect of culture conditions on whole cell OPH activity. Especially, the whole cell OPH activity was significantly affected by the concentration of tetracycline that is an inducer for chaperone GroEL/ES. When cultured at 20°C for 31 h in M9 medium containing 1 mM IPTG, 50 ng/mL tetracycline, and 500 µM CoCl2, the recombinant E. coli exhibited a specific whole cell OPH activity (U/OD600) of ~0.55, which is 2.6-fold higher than that of recombinant E. coli cultured as previously described conditions. In addition, recombinant cells showed adequate storage stability for 1 week with 100% of original response. Finally, the improved activity and adequate stability in the whole cell biocatalyst will contribute to sensitivity, detection time, and stability of a whole cell-based biosensor for the detection of toxic organophosphates.

Releasing intracellular product to prepare whole cell biocatalyst for biosynthesis of Monascus pigments in water-edible oil two-phase system.

Selective releasing intracellular product in Triton X-100 micelle aqueous solution to prepare whole cell biocatalyst is a novel strategy for biosynthesis of Monascus pigments, in which cell suspension culture exhibits some advantages comparing with the corresponding growing cell submerged culture. In the present work, the nonionic surfactant Triton X-100 was successfully replaced by edible plant oils for releasing intracellular Monascus pigments. High concentration of Monascus pigments (with absorbance nearly 710 AU at 470nm in the oil phase, normalized to the aqueous phase volume approximately 142 AU) was achieved by cell suspension culture in peanut oil-water two-phase system. Furthermore, the utilization of edible oil as extractant also fulfills the demand for application of Monascus pigments as natural food colorant.