高产反式-4-羟脯氨酸重组大肠杆菌的构建以及发酵条件优化.docVIP

  高产反式-4-羟脯氨酸重组大肠杆菌的构建 以及发酵条件优化# 刘合栋1,2，袁春伟1,2，张震宇1,2** 5 10 15 20 25 30 35 40 （1. 江南大学工业生物技术教育部重点实验室，江苏 无锡 214122； 2. 江南大学生物工程学院，江苏 无锡 214122） 摘要：脯氨酸 4-羟化酶可直接将游离的脯氨酸的第四位上加羟基生成 4-羟脯氨酸。为了使 脯氨酸 4-羟化酶基因在重组大肠杆菌中得到高表达，根据大肠杆菌密码子偏好性以及 mRNA 二级结构，重新设计合成了脯氨酸 4-羟化酶基因，优化后的脯氨酸 4-羟化酶基因与初始基 因序列比较，其中 139 个碱基通过同义密码子被替换，同时，GC%比从 73.62 %降低到 59.46 %。将优化后的脯氨酸 4-羟化酶基因插入含有色氨酸串联启动子的 pUC19 质粒，构建重组 质粒 pUC19-ptrp2-Hyp，并将重组质粒导入大肠杆菌 BL21(DE3)。在摇瓶水平，重组菌以脯 氨酸为底物发酵 8 h 中可积累 0.492 g/L 的反式-4-羟脯氨酸。在发酵罐水平，利用补料分批 发酵来提高反式-4-羟脯氨酸的产量，以不同的补糖速度流加发酵，结果发现当补糖速度为 0.3 g/L 时，反式-4-羟脯氨酸的产量高达 42.5 g/L，反式-4-羟脯氨酸产率高达 0.924 g/L/h，比 目前所知的最高水平要高 125 %。 关键词：发酵工程；重组大肠杆菌；反式-4-羟脯氨酸；密码子优化；补料策略 中图分类号：Q815 Construction a high yield of trans-4-hydroxyproline recombinant and fermentation optimization LIU Hedong1,2, YUAN Chunwei1,2, ZHANG Zhenyu1,2 (1. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, JiangSu WuXi 214122; 2. School of Biotechnology, Jiangnan University, JiangSu WuXi 214122) Abstract: The proline 4-hydroxylase which catalyzes the conversion of L-proline to 4-hydroxyproline, was expressed in Escherichia coli. To improve expression efficiency of proline 4-hydroxylase in E.coli, the DNA sequence encoding proline 4-hydroxylase was designed and synthesized based on the codon bias of E.coli, the codons encoding 272 amino acids were optimized, in which a total of 139 nucleotides were changed, the G+C ratio was simultaneously decreased from 73.62 to 59.46 %. The optimized proline 4-hydroxylase gene was overexpressed in E.coli BL21(DE3) under a tryptophan tandem promoter in pUC19 vector. At shaking flask level, trans-4-hydroxyproline was accumulated to 0.492 g/L in 8h with L-proline as substrate. For an enhanced trans-4-hydroxyproline production by E.coli BL21(DE3), the effect of constant feeding strategy on trans-4-hydroxyproline production at fermentor level were investigated. With constant feeding approach with F=0.3 g/min, a maximum trans-4-hydroxyproline titer of 42.5 g/L with