甘露醇磷酸化酶基因的敲除对D-甘露醇合成的影响

Effect of Mannitol Phosphorylase Gene Knockout on D-mannitol Synthesis

为了探究甘露醇分解利用途径对甘露醇合成的影响,在重组菌株R1(K-12/pTrc99a-mdh)的基础上,通过CRISPR/Cas9 敲除E.coli PTS 系统中的cmtA、cmtB、mtlA 基因,阻断了E. coli K-12 中甘露醇的分解途径,获得了重组菌株R3(K-12/ΔcmtAΔcmtBmdh^+)和R5(K-12/ΔcmtAΔcmtBΔmtlAmdh^+)。与出发菌株R1 相比,R3 的生长速率没有降低,而R5 的生长速率明显下降。并且R5 在以甘露醇为唯一碳源的培养基上已无法生长,说明cmtA、cmtB、mtlA 三个基因全部敲除后,菌株已无法再利用甘露醇作为碳源进行生长。最后,构建的重组菌株R5,测得MDH 酶活力为258 U/mL,用高效液相色谱对胞外产物进行检测,可检测到少量的甘露醇,为进一步探究大肠杆菌合成甘露醇的调控机制奠定基础。

In order to explore the effect of mannitol decomposition and utilization pathways on mannitol synthesis,the cmtA,cmtB and mtlA genes in Escherichia. coli PTS system were knocked out by CRISPR/Cas9 on the basis of recombinant strain R1(K-12/pTrc99amdh), which blocked the decomposition pathway of mannitol in E. coli K-12,and the recombinant strain R3(K-12/ΔcmtAΔcmtBmdh^+)and R5(K-12/ΔcmtAΔcmtBΔmtlAmdh^+)were obtained. Compared with the original strain R1,the growth rate of R3 did not decrease,but that of R5 decreased significantly. Moreover,R5 did not grow on the medium with mannitol as the sole carbon source,indicating that the strain could no longer use mannitol as the carbon source after all the three genes cmtA,cmtB and mtlA were knocked out. Finally,the recombinant strain R5 was constructed,the activity of MDH enzyme was 258 U/mL,and a small amount of mannitol was detected by high performance liquid chromatography,which laid a foundation for further exploring the regulation mechanism of mannitol synthesis in E. coli.