大肠杆菌PTS^(Ntr)相关基因缺失对L-苏氨酸合成的影响

Effects of Deleting Genes Related to PTS^(Ntr) on L-Threonine Synthesis in Escherichia coli

大肠杆菌的氮磷酸转移酶系统(nitrogen phosphotransferase system,PTSNtr)在其工作过程中会消耗L-苏氨酸合成的前体物质磷酸烯醇式丙酮酸,敲除相关基因或可影响苏氨酸合成。作者利用CRISPR-Cas9系统,分别敲除PTSNtr相关基因ptsP、ptsO和ptsN,构建得到突变株MZ001、MZ002和MZ003。通过对出发菌株和突变菌株测定生长曲线,发现ptsP、ptsO和ptsN的单缺失使菌株培养前期生长迟缓,迟滞期延长,后期快速生长,稳定期生物量积累大于出发菌株。之后在出发菌株和突变菌株中引入含有苏氨酸合成的3个关键酶编码基因thrA*、thrB和thrC以及苏氨酸转运蛋白编码基因rhtC的表达质粒。对比菌株MG1655/pFW01-thrA*BC-rhtC,菌株MZ001/pFW01-thrA*BC-rhtC、MZ002/pFW01-thrA*BC-rhtC和MZ003/pFW01-thrA*BC-rhtC苏氨酸的产量有明显提高,分别为3.805、1.722、3.091 g/L,表明大肠杆菌的PTSNtr相关基因的敲除对提高其L-苏氨酸的合成能力有促进作用。

Phosphoenolpyruvate,the precursor of L-threonine synthesis,is consumed by the nitrogen phosphotransferase system(PTSNtr)of Escherichia coli,so the deletion of genes related to PTSNtr may affect the synthesis of L-threonine.In this study,ptsP,ptsO and ptsN genes encoding the component proteins of PTSNtr in E.coli MG1655 were deleted using CRISPR-Cas9 system,and the corresponding mutants MZ001,MZ002 and MZ003 were constructed.The results showed that the single deletion of ptsP、ptsO or ptsN led to slower growth in the early stage and longer lag period.The mutant strains grew fast in the later stable stage and their biomass accumulation was greater than that of the original strain.Then,the expression plasmid pFW01-thrA*BC-rhtC,which contained thrA*、thrB and thrC encoding threonine synthesis as well as rhtC encoding threonine transporter,was introduced into the original and mutant strains.In comparison with MG1655/pFW01-thrA*BC-rhtC,the L-threonine titer of MZ001/pFW01-thrA*BC-rhtC,MZ002/pFW01-thrA*BC-rhtC and MZ003/pFW01-thrA*BC-rhtC significantly increased by 3.805 g/L,1.722 g/L and 3.091 g/L,respectively.The results showed that the deletion of genes related to PTSNtr of E.coli promoted the synthesis of L-threonine.