3.4. Screening of LPLDH, CPR, and GDH Co-Expressed in E. coli BL21 (DE3)

E. coli BL21 (DE3)/pACYCDuet-1-SceCPR1-EsGDH was successfully constructed, as described previously [8]. The pET28a recombinant plasmid with the gene encoding l-pantolactone dehydrogenase was transferred into E. coli BL21(DE3) competent cells with the recombinant plasmid pACYCDuet-1-SceCPR1-EsGDH, resulting in the strains co-expressing different LPLDH, SceCPR1 and EsGDH.

The genes encoding CduCPR and ZpaCPR were amplified from the plasmids pET28a-CduCPR and pET28a-ZpaCPR, respectively. The PCR program consisted of the following steps: 5 min at 98 °C, 30 cycles of 98 °C (10 s), 59 °C (15 s), and 72 °C (30 s), with a final extension at 72 °C for 5 min. The linear pACYCDuet-1-EsGDH fragment was amplified from the plasmid pACYCDuet-1-SceCPR1-EsGDH by inverse PCR. The corresponding PCR program was as follows: 5 min at 98 °C, 30 cycles of 98 °C (10 s), 58 °C (15 s), and 72 °C (90 s), with a final extension at 72 °C for 5 min. According to the instructions for the ClonExpress^® II One Step Cloning Kit, the genes encoding CduCPR and ZpaCPR were ligated with linear pACYCDuet-1-EsGDH fragments via Exnase II-derived homologous recombination to form recombinant plasmids pACYCDuet-1-CduCPR-EsGDH and pACYCDuet-1-ZpaCPR-EsGDH. Recombinant plasmids pACYCDuet-1-SceCPR1-EsGDH, pACYCDuet-1-CduCPR-EsGDH, and pACYCDuet-1-ZpaCPR-EsGDH were transferred into E. coli BL21(DE3) competent cells with recombinant plasmid pET28a-AmeLPLDH, resulting in the strains co-expressing AmeLPLDH, different CPR, and EsGDH.

Similarly, the gene encoding EsGDH on the plasmid pACYCDuet-1-ZpaCPR-EsGDH was replaced with the gene encoding BmGDH or BsGDH, yielding the recombinant plasmids pACYCDuet-1-ZpaCPR-BmGDH and pACYCDuet-1-ZpaCPR-BsGDH. The recombinant plasmids pACYCDuet-1-ZpaCPR-EsGDH, pACYCDuet-1-ZpaCPR-BmGDH, and pACYCDuet-1-ZpaCPR-BsGDH were transferred into E. coli BL21(DE3) competent cells with the recombinant plasmid pET28a-AmeLPLDH, resulting in the strains co-expressing AmeLPLDH, ZpaCPR, and different GDH. The primers for the co-expression of LPLDH, CPR, and GDH are listed in Table S1.

For each recombinant strain co-expressing LPLDH, CPR, and GDH, single colonies were picked and transferred to 50 mL tubes containing 100 μg/mL kanamycin and 100 μg/mL chloramphenicol in LB medium. The cultures were incubated at 37 °C and 200 rpm for 12 h. When the OD₆₀₀ reached 0.6–0.8, 0.2 mM IPTG was added to initiate induction at 24 °C and 150 rpm for 14 h. Cells were washed twice using 50 mM Tris–HCl buffer (pH 8.0), then harvested by centrifuging at 8000 × g at 4 °C for 10 min. A 12% (w/v) SDS-PAGE was used to visualize the co-expression of LPLDH, CPR, and GDH [33].

To select LPLDH, CPR, and GDH, recombinant E. coli cells as whole-cell biocatalyst were used to catalyze the deracemization of dl-pantolactone into d-pantolactone. The reaction mixture (10 mL) contained 1.0 M dl-pantolactone, 1.5 M d-glucose, 200 g/L wet cells, and 200 mM PBS buffer (pH 7.0). The reaction was conducted at 30 °C and 600 rpm for 24 h, and was terminated by adding an equal volume of 3 M HCl. After that, the mixture was centrifuged and the supernatant was extracted with 5 equivalent volumes of ethyl acetate. The organic phase was harvested by centrifugation at 8000× g for 10 min at room temperature and subsequently dehydrated with anhydrous sodium sulfate. Finally, 200 µL of the resulting dehydrated sample was subjected to GC analysis, as described in Section 3.10.