Efficient synthesis of Ibrutinib chiral intermediate in high space-time yield by recombinant E. coli co-expressing alcohol dehydrogenase and glucose dehydrogenase

Abstract

The production of (S)-N-boc-3-hydroxy piperidine (NBHP) via asymmetric bioreduction of 1-boc-3-piperidinone with reductase is impeded by the need for expensive coenzymes NAD(P)H. In order to regenerate the coenzyme in situ, the gene of alcohol dehydrogenase from Thermoanaerobacter brockii and glucose dehydrogenase from Bacillus subtilis were ligated into the multiple cloning sites of pRSFDuet-1 plasmid to construct the recombinant Escherichia BL21 (DE3) that co-expressing alcohol dehydrogenase and glucose dehydrogenase. Different culture conditions including the medium composition, inducer and pH etc were systematically investigated to improve the enzyme production. The enzyme activity was increased more than 11-fold under optimal culture condition, from 12.7 to 139.8 U L^-1. In the further work, the asymmetric reduction of 1-boc-3-piperidinone by whole cells of recombinant E. coli was systematic optimized to increase the substrate concentration and reaction efficiency. At last, S-NBHP (>99% ee) was prepared at 500 mM substrate concentration without external addition of cofactors. The conversion of S-NBHP reached 96.2% within merely 3 h, corresponding a high space-time yield around 774 g L^-1 d^-1. All these results demonstrated the potential of recombinant E. coli BL21 (DE3) coupled expressing alcohol dehydrogenase and glucose dehydrogenase for efficient synthesis of S-NBHP.

Scheme 1. Asymmetric reduction of 1-boc-3-piperidinone to (S)-N-boc-3-hydroxy piperidine by recombinant E. coli with both TbADH and BsGDH activity.

Fig. 1. Effect of carbon source (A) and its concentration (B) on cell mass (■) and enzyme activity (□). (A). Different carbon source were added into the medium. The recombinant E. coli was cultivated at the medium with different carbon source. After inoculation at 37 °C for 2 h, IPTG (0.1 mM) was added, and then cultivated at 30 °C for 12 h. (B). The recombinant E. coli was cultivated at the medium with different starch concentration. After inoculation at 37 °C for 2 h, IPTG (0.1 mM) was added, and then cultivated at 30 °C for 12 h.

Fig. 2. Effect of nitrogen source (A) and its concentration (B) on cell mass (■) and enzyme activity (□). (A). The recombinant E. coli was cultivated at the medium with different nitrogen source. After inoculation at 37 °C for 2 h, IPTG (0.1 mM) was added, and then cultivated at 30 °C for 12 h. (B). The recombinant E. coli was cultivated at the medium with different nitrogen concentration. After inoculation at 37 °C for 2 h, IPTG (0.1 mM) was added, and then cultivated at 30 °C for 12 h.

Fig. 3. Effect of induction time (A) and induction concentration (B) on cell mass (■) and enzyme activity (□). (A). Medium cultivated at 37 °C, 200 rpm for 0–5 h, then IPTG (0.1 mM) was added, cultivated at 30 °C, 200 rpm for 10 h. (B). Medium cultivated at 37 °C, 200 rpm for 3 h, then different concentrations of induction was added, cultivated at 30 °C, 200 rpm for 10 h.

Fig. 4. Effect of pH (A) and temperature (B) on cell mass (■) and enzyme activity (□). (A). Medium adjusted its initial pH (4.0 ∼ 9.0) cultivated at 37°C, 200 rpm for 3 h, then 0.025 mM IPTG was added, cultivated at 30 °C, 200 rpm for 10 h. (B). Medium cultivated at 37 °C, 200 rpm for 3 h, then 0.025 mM IPTG was added, cultivated in various temperature (20–50 °C), 200 rpm for 10 h, and the enzyme activity was measured in 200 mM sodium phosphate buffer (pH 6.0).

Fig. 5. Enzymatic reduction of 1-boc-3-piperidinone by whole cells cultured in the initial medium (●) or optimized medium (○). A reaction mixture consisting dry cell of 30 g L⁻¹ and 50 mM substrate in 0.2 M phosphate buffer (pH 7.0) was incubated at 30 °C and 200 rpm. Sample was taken at different time and the conversion was determined by GC.

Fig. 6. Effect of temperature on the conversion of N-boc-3-piperidone. The collected cells were resuspended into PBS buffer (pH 8.0, 200 mM). The suspension was incubated at 30 °C, 1000 rpm in thermo shaker for 15 min, and then added the substrate (final concentration of 50 mM) and glucose (final concentration of 75 mM). Methanol of 5% (v/v) was used as the co-solvent. The reaction was carried out at various temperatures (● 30; △40; ■ 50 and ◇ 60 °C) for 10 h. Sample was taken at different time and the conversion was determined by GC.

Fig. 7. Asymmetric reduction of 1-boc-3-piperidinone by E. coli BL21 harboring pRSFDuet-bsgdh-tbadh with different cell concentration. The collected cells were resuspended into PBS buffer (pH 8.0, 200 mM) with various concentration (■ 10; ● 30; and △ 50 g L⁻¹). The suspension was incubated at 30 °C, 200 rpm in a shaker for 15 min, and then added the substrate (final concentration 500 mM) and glucose (final concentration of 750 mM). Methanol of 5% (v/v) was used as the co-solvent. The reaction was carried out at 50 °C for 24 h. Ammonia solution (5 M) was periodical added to the reaction system for neutralizing the produced acid during the reaction. Samples were taken at different time and the conversion was determined by GC.