Efficient synthesis of (S)-phenyl-1,2-ethanediol using xylan as the co-substrate by a multi-enzyme fusion expression system

Abstract:

[Objective] To use xylan as co-substrate to enhance cofactor recycling in chiral catalytic reaction, we constructed a fusion expression system containing (S)-carbonyl reductase (SCRII) from Candida parapsilosis CCTCC M203011, glucose dehydrogenase mutant Ala258Phe (Ala258Phe/GDH) from Bacillus sp. YX-1, and xylanase 2 from Trichoderma reesei Rut C-30 in Escherichia coli BL21. The recombinant E. coli strains efficiently catalyzed 2-hydroxyacetophenone to (S)-phenyl-1,2-ethanediol. [Methods] By adjusting the 3 encoding genes' locations in the pET-28a, 2 recombinant plasmids pET-SCRII-A258F-XYN2 and pET-A258F-SCRII-XYN2 were constructed by using the overlap extension PCR technology. The optimal temperature, pH value and the best ratio of 2-hydroxyacetophenone and co-substrate xylan for catalyzing (S)-phenyl-1,2-ethanediol by the recombinant E. coli/pET-SCRII-A258F-XYN2 and E. coli/pET-A258F-SCRII-XYN2 were determined. [Results] Through the optimization of pH, temperature and the ratios between substrate and co-substrate, the recombinant E. coli/pET-SCRII-A258F-XYN2 produced (S)-phenyl-1,2-ethanediol with a yield of 98.8% (W/W) under the optimal conditions:35℃, pH 7.0 and a 1:1 substrate-co-substrate ratio, while the recombinant E. coli/pET-A258F-SCRII-XYN2 produced (S)-phenyl-1,2-ethanediol with a yield of 95.6% (W/W) under the optimal conditions:35℃, pH 7.0 and a 2:1 substrate-co-substrate ratio. The two recombinant strains catalyzed (S)-phenyl-1,2-ethanediol with an optical purity >99%. [Conclusion] In the fusion expression system containing three enzymes, xylanase and glucose dehydrogenase mutant mediated cofactor regeneration was introduced into asymmetric biosynthesis reactions, which efficiency improved chiral biotransformation. This work supplied a more solid foundation by using the naturally abundant xylan for chiral catalysis.