Abstract: 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/pTrc99a-mdh）,which blocked the decomposition pathway of mannitol in E. coli K-12,and the recombinant strain R₃（K-12/∆cmtA∆cmtBmdh⁺）and R₅（K-12/∆cmtA∆cmtB∆mtlAmdh⁺）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.

Key words: gene knockout, mannitol, mannitol dehydrogenase, NADH