Ene-reductase screening
The first stage of the study was the selection of enzymes capable of reducing C=C double bond in (R)-(–)-massoia lactone (1) (Fig. 1), obtained by extraction from the Cryptocaria massoia tree bark. The regeneration of the NADPH cofactor was performed using a glucose dehydrogenase from Bacillus megaterium, with glucose as a co-substrate21.
OYE-mediated reduction of massoia lactone (1) to (R)-(+)-δ-decalactone (2).
Selected enzymes of microbial and plant origin were obtained using the Escherichia coli expression system. Each of the 13 selected ene-reductases was isolated, purified, and tested for biotransformation of (R)-(–)-massoia lactone (1) to (R)-(+)-δ-decalactone (2). The degree of conversion was checked after 72 h (Fig. 2).
Results of massoia lactone (1) conversion by selected ene-reductases after 72 h (reaction conditions: 10 µL of 500 mM massoia lactone, 10 µL of 10 mM NADP+ solution, 20 µL of 1 M glucose solution, and 10 µL of GDH (5 mg/mL), ene-reductase: 100 µL (OYE1—50 µL, OYE2, OYE2.6, OYE3 and NemA—40 µL), 50 mM phosphate buffer pH 7–1 mL).
Of the 13 ene-reductases tested, five of them catalyzed the total reduction of lactone 1 to the desired product 2. In a further screening stage, it was decided to study the degree of substrate 1 conversion using tenfold less of selected enzymes (OYE1, OYE2, OYE3, NemA and YqjM) in the reaction mixture, and the biotransformation products were extracted after 24 h (Fig. 3).
Results of massoia lactone (1) conversion by selected ene-reductases after 24 h (reaction conditions: 10 µL of 500 mM massoia lactone, 10 µL of 10 mM NADP+ solution, 20 µL of 1 M glucose solution, and 10 µL of GDH (5 mg/mL), ene-reductases: OYE1—5 µL, OYE2, OYE3 and NemA—4 µL, YqjM—10 µL, 50 mM phosphate buffer pH 7–1 mL).
The highest conversion (> 99%) of (R)-(–)-massoia lactone (1) was achieved when OYE3 was used as a biocatalyst, negligible lower conversion (98%) was observed with YqjM enzyme. Considering the obtained results, for further study of massoia lactone reduction OYE3 was chosen as a biocatalyst.
The substrate spectrum and stereoselectivity of yeast origin OYEs, among them OYE3, have been extensively studied in the last decade10,17,21,22. The advantages of using OYE-catalyzed hydrogenation for the synthesis of Active Pharmaceutical Ingredients (APIs) and fragrances have been demonstrated in several studies23,24. The OYE3 was obtained and purified for the first time by Niino et al. from yeast S. cerevisiae using E. coli expression system. The studies performed by his research group indicated that OYE3 (44,920 Da) has 73% similarity of DNA sequence and 89% similarity of amino acid sequence to OYE2. Despite the similarities, both enzymes exhibit different activities—OYE3 showed higher reducing activity25. Tasnádi et al. investigated various ene-reductases (among them OYE1–3, YqjM and OPR1), which have been studied for the bioreduction of cinnamic-ester derivatives. They established that OYE3 has ability to accept larger substrates due to the presence of serine (S297) in the binding pocket, whereas OYE1 and OYE2 have smaller binding pocket caused by the presence of phenylalanine (F296)26.
The use of isolated enzyme for the biotransformation of massoia lactone has significant advantages, because it avoids undesirable side reactions and ensures high yields. However, it requires many, sometimes expensive unit operations, such as subjecting cells to a disintegration process, and then purifying the desired enzymes by using chromatographic techniques or molecular filtration.
Reduction of (R)-(–)-massoia lactone (1) in batch system with resting cells
The use of resting cells is an attractive solution, because this method requires the least number of unit operations. Additionally, there is no necessity to add to the reaction environment a cofactor and its regeneration system components, which are essential for an enzyme activity.
To investigate whether resting cells also catalyze the biotransformation of massoia lactone (1), reactions were carried out using different amount of E. coli BL21(DE3)/pET30a-OYE3 biomass (30 and 60 mg cells/mL reaction mixture) suspended in phosphate buffer (50 mM, pH 7.0) and different substrate 1 concentration (3 and 6 mM). Chromatographic analysis of the post-reaction mixture (after 24 h) showed the presence of only (+)-δ-decalactone ((+)-2) regardless of the applied biotransformation conditions. The structure of product was confirmed by 1H NMR and 13C NMR.
Reduction of (R)-(–)-massoia lactone (1) in batch system with immobilized cells
In a further study, the strategy of whole-cell immobilization was employed to enhance the reusability of the biocatalytic system. Commonly applied methods include covalent linking to solid matrices, or entrapment and encapsulation in polymeric networks. Among these approaches, one of the most convenient methods for cells immobilization is encapsulation in alginate beads. Their mild gelation condition, high porosity and inert aqueous matrix help preserve the properties of the encapsulated biomass. However, dissolution of alginate beads was observed when the most convenient phosphate buffer (50 mM, pH 7.0) for OYE3 reduction was used as a reaction medium. In order to find the suitable conditions for immobilized E. coli BL21(DE3)/pET30a-OYE3 cells for the bioreduction of (R)-(–)-massoia lactone (1) to (R)-(+)-δ-decalactone (2), several approaches were applied. In the first one, biomass was suspended in either Tris–HCl (50 mM, pH 7.0) or sodium acetate buffer (20 mM, pH 6.0), immobilized using 4% solution of sodium alginate. The alginate gel beads (containing approximately 200 mg of cells) thus obtained were used as a biocatalyst in the wet and dried form.
The dissolution of alginate beads was not observed, when these buffers were used as a reaction environment. However, the results of biotransformation indicate the presence of desired product 2 merely when wet beads in Tris–HCl buffer were employed. The maximum conversion of (R)-(–)-massoia lactone (1) to 2 (85%) was achievedafter 96 h of reaction. Complete lack of the ene-reductase activity was observed when sodium acetate buffer was used.
Although dried alginate beads with entrapped microbial biomass and enzymes were successfully applied in other biotransformation processes16,27, in this work a complete loss of reduction activity of immobilized E. coli cells overexpressing the OYE3 enzyme was observed. Considering the obtained results, it was decided to continue experiments using immobilized E. coli cells in the form of wet beads in Tris–HCl buffer. To ensure better exchange of substrate 1 and product 2 between the entrapped cells and the reaction environment, entrapment was performed using sodium alginate solution with twice lower concentration (2%). Additionally, to check the impact of glucose on the reduction of massoia lactone (1) to (+)-δ-decalactone (2), glucose (0.2% w/v) was added to the reaction mixture.
The results presented in Fig. 4 (case C and D) show that the addition of glucose definitely improves massoia lactone (1) reduction process, probably due to the fact that it is an essential substrate for NADP+ regeneration (Fig. 1) or the addition of glucose probably led to a metabolic boost affecting positively the efficiency of cofactor regeneration. In these cases, the complete transformation of substrate 1 was determined in the first sample collected after 12 h of biotransformation. Additionally, the lower degree of entrapment accelerated the biotransformation process.
Time-course of the reduction of massoia lactone (1) with immobilized cells of E. coli BL21(DE3)/pET30a-OYE3 in batch mode. (A) 200 mg immobilized biomass without glucose, (B) 400 mg immobilized biomass without glucose, (C) 200 mg immobilized biomass with glucose, (D) 400 mg immobilized biomass with glucose.
The immobilization of biocatalyst was also performed using agar–agar for entrapment. The analysis indicated that the complete conversion of (R)-(–)-massoia lactone (1) to δ-decalactone (2) was achieved after 12 h of reaction using agar–agar cubes with immobilized biomass of E. coli BL21(DE3)/pET30a-OYE3. The investigation on OYE3 immobilization was conducted by Tentori et al., where OYE3 was immobilized both by covalent binding on glyoxyl-agarose (GA), and by affinity-based adsorption on EziGTM particles17. The OYE3/GDH-EziG (co-immobilized OYE3 and GDH) exhibit lower bioreduction activity against α-methyl-trans-cinnamaldehyde than OYE3-GA, which was probably related to the loss of immobilized GDH activity during the biotransformation process.
Both the biocatalyst in the form of Ca2+-alginate beads and agar–agar cubes showed similar reducing activity against substrate (1). Considering the convenience of obtaining the biocatalyst in the form of Ca2+-alginate spheres, it was decided to continue the research by immobilizing E. coli BL21(DE3)/pET30a-OYE3 (co-expressing OYE3 and GDH) using this technique.
Next experiments were performed in batch conditions with different massoia lactone concentration. Beads with entrapped E. coli BL21(DE3)/pET30a-OYE3 cells containing approximately 200 mg of wet biomass were introduced into 20 mL glass vials and a solution consisting of a mixture of Tris–HCl buffer, glucose (0.2% and 0.5% w/v) and substrate 1 (3–40 mM) in a final volume of 5 mL was added. The Fig. 5 shows results of 4-h reaction where the highest conversion (> 99%) was achieved in the batch with the 3 mM concentration of substrate 1), whereas the use of a 10 mM massoia lactone (1) solution reduced the conversion by 5 times.
Conversion of massoia lactone (1) with immobilized cells of E. coli BL21(DE3)/pET30a-OYE3 in batch mode with different concentration of substrate 1 (4 h reaction time).
Flow biotransformation with immobilized cells
The reduction process of massoia lactone (1) was also tested under continuous-flow conditions, by the application of a packed-bed bioreactor. Alginate beads with immobilized E. coli BL21(DE3)/pET30a-OYE3 cells (3.5 g) were loaded onto a glass column (i.d. 15 mm, length 3 cm). A first set of experiments was carried out by simply feeding the packed bed bioreactor with massoia lactone (1) (3 mM) and glucose (0.5% w/v) solution in Tris–HCl buffer at different flow rates; however, no biotransformation was observed, regardless of the set flow. Therefore, it was decided to supply oxygen to the reaction environment by a segmented gas–liquid flow. Providing oxygen to the immobilized cells supported their vital functions, enabling the reduction of massoia lactone (1). The gas phase and the liquid phase were merged in a T-junction (air flow: 100 µL/min, liquid flow: 100 µL/min, residence time: 25 min), allowing to generate air–buffer solution segments in the flow stream entering the packed-bed reactor (Fig. 6). Flow biotransformation of substrate 1 was also conducted using cells entrapped in agar–agar as biocatalysts.
Scheme of packed-bed bioreactor with the segmented gas–liquid flow applied in experiment.
The application of flow system avoids problems related to substrate and product inhibition impact on immobilized biocatalyst; however, the complete conversion to desired product 2 was not achieved and the reduction activity exhibited by immobilized E. coli cells decreased during semi-continuous operation (Fig. 7). This could be due to partial damage of the immobilized system, the NADP+ cofactor that participates in the reduction system may be lost by cells damaged by shear stress occurring in flow reactors16.
Conversion of massoia lactone (1) with immobilized cells of E. coli BL21(DE3)/pET30a-OYE3 in semi-continuous flow system with the use of segmented gas–liquid flow.
Flow biotransformation with cell lysate
Finally, it was decided to use the biocatalyst in the form of the E. coli BL21 (DE3) pET30a-OYE3 cell lysate obtained by sonication. The use of cell lysate allows the cell-reaction environment barrier to be bypassed, ensuring better access of the enzyme to the substrate. The reduction of massoia lactone (1) was conducted in flow reactor with a membrane system. The semi-cellulose membrane constituted a semi-permeable barrier, retaining the enzymes contained in the cell lysate and letting a biotransformation product 2 (δ-decalactone) passthrough the system. The membrane could retain both OYE3 and GDH, allowing the continuous use of the whole recycling system and saving NADP+ that flew through reactor with glucose and the substrate 1 solution (Fig. 8).
Scheme of reactor system with a semi-permeable membrane between cell lysate and massoia lactone (1) solutions.
The solution of 10 mM massoia lactone (1) was pumped at a constant flow rate of 100 µL/min in thermostatic condition through a semi-cellulose membrane placed in the membrane reactor containing the solution of lysate and cofactor regeneration system. The residence time was 120 min. In the analysed samples the presence of (R)-(+)-δ-decalactone (2) was detected. The outlet conversion was constant (> 99%), no decrease in the reducing activity of the catalyst was observed until the end of the flow process (5 h). The space–time yield (STY) for continuous-flow reduction with lysate was calculated as below:
$${STY}_{flow}=frac{{n}_{reagent}cdot C}{{V}_{lysate}cdot tau }=frac{0.3cdot 99}{0.002cdot 2}=7.425 mathrm{M }{mathrm{h}}^{-1} {mathrm{L}}^{-1}$$
where nreagent (mmol)—amount of reagent; C (%)—conversion of the reagent into the desired product determined by GC/MS analysis; Vlysate (L)—volume of lysate solution; τ (h)—residence time28
