多酶融合表达体系以木聚糖为辅助底物高效合成(S)-苯乙二醇

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

【目的】利用木聚糖为辅助底物加强手性催化反应中的辅酶循环,构建来源于近平滑假丝酵母(Candida parapsilosis) CCTCC M203011的(S)-羰基还原酶Ⅱ(SCRⅡ)、枯草芽孢杆菌(Bacillus sp.) YX-1葡萄糖脱氢酶突变体Ala258Phe/GDH和里氏木霉(Trichoderma reesei)RutC-30木聚糖酶(XYN2)在大肠杆菌(Escherichia coli) BL21(DE3)中的融合表达体系,高效合成(S)-苯乙二醇。【方法】调节3种酶编码基因在pET-28a载体上的位置,运用重叠延伸PCR技术,构建了E.coli/pET-SCRⅡ-A258F-XYN2和E. coli/pET-A258F-SCRⅡ-XYN2两种重组菌,研究了其合成(S)-苯乙二醇的最适反应条件。【结果】重组菌株E.coli/pET-SCRⅡ-A258F-XYN2在底物2-羟基苯乙酮与辅助底物木聚糖的比例为1:1、35℃、pH为7.0条件下,(S)-苯乙二醇的产率达98.8%(W/W);而重组菌株E. coli/pET-A258F-SCRⅡ-XYN2在底物与辅助底物的比例为2:1、35℃、pH为7.0条件下,(S)-苯乙二醇的产率达95.6%(W/W),两者合成产物的光学纯度均>99%。【结论】通过构建3种酶的融合表达体系,成功将木聚糖酶和葡萄糖脱氢酶突变体介导的辅酶再生循环体系引入不对称生物合成反应,提高了手性转化效率,为将大自然中丰富的木聚糖用于手性催化奠定了较扎实的研究基础。

[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.