基因重组菌质粒稳定性的提高与苯丙氨酸发酵的研究

Stability Improvement of Plasmid Harboured in Genetically Engineered Strain and Production of Phenylalanine

本研究用具有苯丙氨酸生产抗反馈抑制基因pheAFR、aroYFR及温度敏感型阻遏基因CI857的质粒pSY130-14和具有分配机能的低拷贝质粒pSY16,重组构建了具有苯丙氨酸生产基因系统的质粒pSY200-14,然后使其转化到大肠杆菌AT2471中,育成了基因重组菌株AT2471/pSY200-14。试验表明,该菌株质粒稳定性比原菌株AT2471/pSY130-14有较大的提高,当存在选择压时,在30—42℃范围内维持100%的高稳定性。应用此重组菌株,在2.5L通气搅拌罐进行发酵试验,在搅拌转速850rpm,通气速率1.0vvm,38.5℃和pH7.0的条件下,发酵48h苯丙氨酸生成量达14.2g/L,比原株增产11.8%。

In this study, a new plasmid pSY200-14 was constructed by generecombination technology, which has a genetic system of phanylalanine production, by using plasmid pSY130-14 and pSY16. The pSY130-14 contains pheA^(FR) and aroF^(FR), the feedback inhibition resistant phenylalanine-production genes, and a temperature-sensitive repressor cI(857), whereas pSY16 possesses a partition system and is a low copy plasmid. Subsequent Experiments have shown that the recombinant AT2471/pSY200-14 constructed is more stable than original strain AT2471/pSY130-14, in other words, the former kept the stability as high as 100% under selective pressure in 30--42℃. In a 2.5L jar fermentor, the production of phenylalanine after 48h cultivation reached 14.2g/L that was 11.8% higher than old strain under the conditions of impeller speed 850 rpm, aeration rate 1.0 vvm, temperature 38.5℃ and pH7.0.