摘要
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R_1(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R_3(K-12/?cmtA?cmtBmdh~+)和R_5(K-12/?cmtA?cmtB?mtlAmdh~+)。与出发菌株R_1相比,R_3的生长速率没有降低,而R_5的生长速率明显下降。并且R_5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R_5,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 R_1(K-12/pTrc99 amdh),which blocked the decomposition pathway of mannitol in E. coli K-12,and the recombinant strain R_3(K-12/?cmtA?cmtBmdh~+)and R_5(K-12/?cmtA?cmtB?mtlAmdh~+)were obtained. Compared with the original strain R_1,the growth rate of R_3 did not decrease,but that of R_5 decreased significantly. Moreover,R_5 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 R_5 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.
引文
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