利用Red同源重组技术构建产L-苏氨酸的基因工程菌

利用Red重组技术构建不同基因突变的L-苏氨酸工程菌大肠杆菌ITHR，研究单敲除metA、ilvA和双敲除metA、ilvA基因后对L-苏氨酸积累的影响。应用质粒pKD46介导的Red同源重组系统，通过第一次同源重组将拟敲除基因替换为氯霉素抗性基因，再通过重组酶在FRT位点发生第二次同源重组，消除抗性基因，成功敲除了菌株ITHR体内苏氨酸合成的代谢旁路途径中的metA和ilvA基因，构建了三株不同的基因突变株。将携带苏氨酸操纵子的工程质粒pWYE065电转化入敲除不同基因的突变株中，构建基因工程菌。经5 L发酵罐发酵产酸实验，未敲除任何基因的菌株ITHR/pWYE065 L-苏氨酸的产量为5.55±0.51 g/L，metA基因单敲除菌株ITHR△metA/pWYE065 L-苏氨酸产量为9.77±1.83 g/L，ilvA基因单敲除菌株ITHR△ilvA/pWYE065 L-苏氨酸产量为8.65±1.42 g/L，同时敲除ilvA和metA基因的菌株ITHR△metA△ilvA/pWYE065 L-苏氨酸的产量增加到13.6±1.14 g/L。通过敲除L-苏氨酸的旁路代谢途径中的关键酶的基因，可以增强L 苏氨酸积累的效果，为L-苏氨酸工程菌的进一步改造奠定了基础。

Construction of Genetic Engineering Strains for L-threonine Production by Red Recombination

The threonine genetic engineering strains were constructed by a strategy of cut off branch metabolic pathways and overexpression thr operon in E. coli ITHR. The metA and ilvA genes were knocked out by Red-recombination systems and E. coli ITHR ΔmetA, ITHR ΔilvA and ITHR ΔmetA ΔilvA were constructed respectively. The pWYE065 containing thr operon were transformed into three mutant strains by electroporation. Red-batch cultures of genetic engineering strains were carried out in 5 L fermentors and the threonine concentration was determined by HPLC. The results showed that E. coli ITHR carrying pWYE065 could accumulated 5. 55 ±0.51 g/L threonine. When metA and ilvA were knocked out respectively, the threonine productions reached 9. 77 ± 1. 83 g/L and 8. 65 ± 1. 42 g/L. Both metA and ilvA were knocked out simultaneously, the L-threonine production were increased to 13. 6 ± 1. 14 g/L. The productivity of L-threonine by E. coli ITHR can be enhanced through the knocking out the gene of the key enzymes in branch metabolic pathways, including the methionine and isoleucine synthesis pathway.