脯氨酸-4-羟化酶在大肠杆菌中的密码子优化表达及对反式-4-羟脯氨酸生物合成的作用

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,通过调整大肠杆菌密码子偏好性以及mRNA二级结构,使得脯氨酸-4-羟化酶基因得到优化。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p UC19质粒,构建重组质粒p UC19-ptrp2-Hyp,并导入大肠杆菌BL21(DE3)中。在摇瓶水平,重组菌以L-脯氨酸为底物发酵8 h,可积累(0.492±0.034)g/L的反式-4-羟脯氨酸。在发酵罐水平,通过补料分批发酵来提高反式-4-羟脯氨酸的产量,当补糖速率为18 g/h时,反式-4-羟脯氨酸的产量高达42.5 g/L,反式-4-羟脯氨酸产率为0.966 g/(L·h)。

Expression of codon-optimized proline-4-hydroxylase in Escherichia coli and its effect on trans-4-hydroxyproline biosynthesis

To improve expression of proline 4-hydroxylase in Escherichia coli,the DNA sequence encoding proline 4-hydroxylase was adjusted based on the codon bias of E-coli and the mRNA secondary structure-The optimized proline-4-hydroxylase gene was overexpressed in E-coli BL21 （ DE3 ） under a tryptophan tandem promoter in pUC19 vector-In shaking flask culture, trans-4-hydroxyproline was accumulated to （ 0.492± 0.034 ） g/L in 8 h 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 in a fermentor was studied-With constant feeding rate at 18 g/h,a maximum trans-4-hydroxyproline titer of 42-5 g/L with a high trans-4-hydroxyproline productivity （0.966 g/（L·h）） was achieved.