大肠杆菌trpBA和serA基因的串联表达

Co-expression of trpBA and serA Genes in Escherichia coli

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37 kD (PGDH)、29 kD(色氨酸合成酶的α亚基)、44 kD(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。

Abstract:

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, respectively. 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.