大肠杆菌trpBA和serA基因的串联表达  

Co-expression of trpBA and serA Genes in Escherichia coli

作　　者：张绪梅[1,2] 郭长江[1] 刘云[3] 徐琪寿[3] 

机构地区：[1]军事医学科学院卫生学环境医学研究所,天津300050 [2]天津医科大学公共卫生学院,天津300070 [3]军事医学科学院放射医学研究所,北京100850

出　　处：《微生物学通报》2009年第1期2-8,共7页Microbiology China

基　　金：天津市科技攻关项目(No.033182711);国家自然科学基金资助项目(No.30300010)

摘　　要：大肠杆菌trpBA基因编码的色氨酸合成酶（tryptophan synthetase，TSase）是色氨酸合成的关键酶；serA基因编码的磷酸甘油酸脱氢酶（D-3-phosphoglycerate-dehydrogenase，PGDH）为L-丝氨酸合成（色氨酸合成的底物）的关键酶。为了通过基因工程手段来增加色氨酸的产量，在利用高效的原核表达载体pET22b（＋）分别对trpBA和serA基因克隆表达的基础上，采用PCR方法扩增了抗反馈抑制的serA和trpBA基因，将两基因串联于pET22b（＋）载体上，共构建了4种方式的串联质粒，实现了2种蛋白酶在大肠杆菌中的共表达。聚丙烯酰胺电泳分析显示，ABA—I重组菌株在37kD（PGDH）、29kD（色氨酸合成酶的α,亚基）、44kD（β亚基）处均有明显的蛋白表达带。4种串联表达质粒重组菌的TSase酶活性，分别比含空载体菌相应酶的活性提高2～4倍，PGDH酶活性分别提高约2．1～3．6倍。经摇瓶发酵实验表明酶活性较高的ABA—I菌株色氨酸合成量亦最高，约为对照菌株的20．2倍。Escherichia coli trpBA-encoded tryptophan synthetase （TSase） and serA-encoded D-3-pho- sphoglycerate dehydrogenase （PGDH） are key enzymes in tryptophan and serine biosynthesis pathway, re spectively. In order to improve bio-production of tryptophan through bioengineering means, a feedback inhibition resistant serA gene was cloned by PCR and co-expressed with trpBA gene, which was cloned and expressed before. Four recombinant plasmids were constructed successfully in the recombinant strains. SDS-PAGE （sodium dodecyl sulfate polyacrylamide gel electrophoresis） showed target protein products of 37 kD （PGDH）, 29 kD （TSase α subunit）, 44 kD （TSase β subunit）. The enzyme activity analysis indicated the specific activities of TSase was increased by 2-4-fold, and that of PGDH was increased by 2.1-3.6-fold, compared to the control. High enzyme activities could lead to high tryptophan production by the shake flask fermentation. The amount of tryptophan biosynthesis in ABA-I strain was increased by 20.2 folds compared with that of the host strains.

关 键 词：色氨酸 大肠杆菌 serA基因 trpBA基因 串联表达 

分 类 号：Q78[生物学—分子生物学]