摘要
目的:改造大肠杆菌苯丙氨酸生物合成的中心代谢途径,优化关键酶基因pheA、aroF、ppsA、tktA的协同表达,进一步提高苯丙氨酸产量。方法:构建重组质粒pZE12-AFPT,鉴定后通过SDS-PAGE观察其蛋白表达量,并转入缺陷菌大肠杆菌MGΔ中构建工程菌,发酵培养后测量苯丙氨酸产量,与本室保存的重组质粒MGΔpZE12-AF做对比;构建重组质粒pZE21-AF和pZA31-PT,将后者转入感受态pZE12-AF和pZE21-AF中,得到双抗性质粒,并比较转化前后苯丙氨酸的产量。结果:工程菌MGΔpZE12-AFPT的苯丙氨酸产量比对照菌株MGΔpZE12-AF提高了近1.6倍,并且实现了4个串联基因的协同表达;质粒pZA31-PT转入pZE12-AF和pZE21-AF后,苯丙氨酸产量比原质粒pZE12-AF和pZE21-AF分别提高了近0.6倍和2.8倍。结论:实现了4个关键酶基因的串联表达,改造了苯丙氨酸的生物合成途径,使得苯丙氨酸产量有所提高,为进一步得到其高产菌株奠定了基础。
Objective： To improve the production of phenylalanine through reforming the center metabolic pathways of phenylalanine biosynthesis in E.coli strain and optimizing the protein expression of four key genes pheA, aroF, ppsA and tktA. Methods： We constructed one recombinant plasmid pZE12-AFPT and observed its protein expression by SDS-PAGE after the verification. Then, it was transformed into an auxotrophic E.coli strain MGA to get an engineering E.coli strain and the production of phenylalanine was measured after fermented. Finally, it was compared against recombinant plasmid MGApZE12-AF which saved in our lab. Constructing two recombinant plas- mids pZE21-AF and pZA31-PT, we transformed the latter into pZE12-AF and pZE21-AF to get new plasmids with double resistance, then, compared the phenylalanine production before and after transforming. Results： L-phe- nylalanine yield of the engineered strain MGApZE12-AFPT were almost 1.6 times compared with the engineered strain MGApZE12-AF, and it achieved by coordinated tandem expression of the four genes. The yield of L-phenyl- alanine enhanced almost 0.6 and 2.8 times compared with the original plasmids pZE12-AF and pZE21-AF after the plasmid pZA31-PT transformed into. Conclusion： This experiment accomplished tandem expression of the four genes and improved the biosynthetic pathway of phenylalanine. The yield of L-phenylalanine was enhanced which provided an approach to get the high-yielding strains.
出处
《生物技术通讯》
CAS
2012年第4期488-492,共5页
Letters in Biotechnology
基金
军事医学科学院创新基金(9902502)
关键词
苯丙氨酸
大肠杆菌
串联表达
甘油
phenylalanine
E.coli
tandem expression
glycerine
