亮氨酸脱氢酶与葡萄糖脱氢酶高效共表达制备L-叔亮氨酸

High efficient co-expression of leucine dehydrogenase and glucose dehydrogenase in Escherichia coli

摘要:

[目的] 通过不同双基因共表达策略对亮氨酸脱氢酶和葡萄糖脱氢酶基因在大肠杆菌中表达影响的研究，获得具有高辅酶再生效率的双酶共表达重组生物催化剂，实现L-叔亮氨酸“一锅法”高效不对称合成。[方法] 以来自于蜡状芽孢杆菌（Bacillus cereus）的亮氨酸脱氢酶（LDH）和来自芽孢菌属（Bacillussp.）的葡萄糖脱氢酶（GDH）为模板，考察单质粒共表达，双质粒共表达和融合表达等3种共表达策略对重组细胞中亮氨酸脱氢酶和葡萄糖脱氢酶活的影响，比较不同酶活比例和不同催化剂形式对三甲基丙酮酸不对称还原制备L-叔亮氨酸效率的影响。[结果] 研究发现不同共表达策略对亮氨酸脱氢酶和葡萄糖脱氢酶的影响存在明显差异。亮氨酸脱氢酶在不同策略下均能够正常表达，而葡萄糖脱氢酶在融合表达时没有活力，当C端含有组氨酸标签时，表达蛋白活性低。通过表达优化，获得3株亮氨酸脱氢酶和葡萄糖脱氢酶高效表达且具有不同酶活比例的重组菌。比较粗酶液和全细胞形式下的催化效率，发现酶活比例及催化剂形式对不对称还原反应效率具有重要影响。确定单质粒串联表达C端不含His标签重组菌E.coli BL21/pET28a-L-SD-AS-G为最佳催化剂，以粗酶液进行转化时，完全转化0.5 mol/L底物所需菌体量为15 g/L，辅酶量为0.1 mmol/L。[结论] 采用单质粒共表达策略，成功构建出1株具有较高亮氨酸脱氢酶和葡萄糖脱氢酶活性的重组菌，实现高效催化TMP合成L-Tle。

Abstract:

[Objective] Different co-expression strategies to express leucine dehydrogenase and glucose dehydrogenase in E. coli were done to observe the effect of expression of different enzyme. A recombinant strain with two high enzyme activities was built for efficiently asymmetric synthesis of L-tert-leucine.[Methods] The leucine dehydrogenase (ldh) from Bacillus cereus and glucose dehydrogenase (gdh) from Bacillus sp. were co-expressed by three different strategies, including co-expressing two genes in single vector, co-expressing two genes in two vectors and expressing fusion protein. The catalytic efficiencies of recombinant strains with different enzyme activity ratio in different modes of biocatalyst were compared to produce L-tert-leucine from its corresponding α-keto acids.[Results] Different co-expression strategies displayed a slight impact on leucine dehydrogenase expression, whereas, a greater impact on glucose dehydrogenase. All the activity of leucine dehydrogenase was normally expressed, but the fusion proteins lost the activity of glucose dehydrogenase. Besides, the activity of glucose dehydrogenase was also totally inhibited when the 6-histidine tag was fused at C termini, which indicated the additional 6-histidine tag considerately depressed the glucose dehydrogenase activity. After optimization of expression, three recombinant strains exhibiting high enzyme activity and different enzyme activity ratio were used to synthesis L-tert-leucine in the mode of cell-free extracts and whole-cell. Result displayed a great influence on the catalytic efficiencies resulted from the mode of catalyst and enzyme activity. When the cell-free crude culture broth of E. coli BL21/pET28a-L-SD-AS-G coexpressing two genes in single vector was used as biocatalyst, 15 g/L cell loading and 0.1 mmol/L NAD⁺ were enough to completely transform 0.5 mol/L trimethylpyruvate into L-tert-leucine.[Conclusion] The recombinant strain with high activities of leucine dehydrogenase and glucose dehydrogenase was achieved by co-expressing two genes in single vector without histidine tag in E. coli and L-tert-leucine was efficiently produced with this recombinant strain.