产L-苯丙氨酸工程菌的构建及不同碳源对其产量的影响

Construction of L-phenylalanine-producing strain and effect of different carbon sources on yield of L-phenylalanine

目的构建L-苯丙氨酸高产工程菌株,并比较甘油和葡萄糖两种碳源对工程菌产L-苯丙氨酸的影响。方法 PCR克隆glpK基因,改变其核糖体结合位点(RBS)序列,分别与载体pZE12-AF连接,得到pZE12-AF-RBSⅠ-glpK和pZE12-AF-RBSⅡ-glpK两个重组质粒,再将它们分别导入E.coli MG△观察其SDS-PAGE蛋白电泳;采用HPLC法检测在不同碳源中L-苯丙氨酸的产量。结果工程菌E.coli MG△pZE12-AF-RBSⅠ-glpK中甘油激酶表达量较工程菌E.coli MG△pZE12-AF-RBSⅡ-glpK有所提高;以甘油为碳源时,工程菌E.coli MG△pZE12-AF-RBSⅡ-glpK的L-苯丙氨酸产量分别是工程菌E.coli MG△pZE12-AF-RBSⅠ-glpK的14.16倍,工程菌E.coli MG△pZE12-AF的3.18倍,E.coli MG△的63.31倍。以甘油为碳源时,工程菌苯丙氨酸产量高于以葡萄糖为碳源时的苯丙氨酸产量。结论 RBS的改变和glpK基因的表达有利于苯丙氨酸产量的提高,甘油较葡萄糖更适宜作为工程菌生长的碳源。

Objective To construct the L-phenylalanine-producing engineering strains and observe the effect of glucose and glycerol, when used as carbon source, on the growth of recombination strains. Methods GlpK gene was amplified by PCR and changes were made in its ribosome binding site（RBS） sequence, respectively before both were attached to pZE12- AF vectors. Two recombinant plasmids, pZE12-AF--RBS Ⅰ -glpK and pZE12-AF-RBS Ⅱ -glpK, were obtained. Then, they were transformed into Esherichia coli MGA separately for the protein expression by SDS-PAGE. The effect on the yield of L-phenylalanine was assayed by HPLC after fermentation. Results The expression level of glpk gene in the engineering strain of E. coli MGApZE12-AF- RBS Ⅰ -glpK was higher than that in E. coli MGApZE12-AF-RBS Ⅱ-glpK strain. When glycerol was used as carbon source, L-phenylalanine in the engineering strain E. coli MGApZE12-AFRBS ll-glpK was 14.16 times the production of the engineering strain E. coli MGApZE12-RBS-AF, 3.18 times that of the engineering strain E. coli MGApZE12-AF and 63.31 times that of the native strain E. coli MGA. The L-phenylalanine productivity of all the strains was higher when glycerol was used as the carbon source than when glucose was used. Conclusion Changes of RBS sequence and the expression of glpK gene could increase the output of L-phenylalanine in the engineering strain, and glycerol is a better carbon source for recombination strains than glucose.