The Study Of D-Mannitol Synthesis And Decomposition Related Enzymes And Pathway In Escherichia Coli

The mannitol,with good stability,is a kind of functional sugar alcohol,which has many advantages,such as pressure resistance,anti-adhesive,non-hygroscopicity,et,al.Mannitol has a wide range of applications in medicine,food and chemical industry.Compared to other methods,biotransformation has many advantages such as mild conditions,fewer by-products and higher yields.The synthesis and decomposition pathway of mannitol in E.coli K-12 was studied and the expression and transformation conditions of the key enzymes were explored in this study;The pathway of mannitol decomposition were blocked up,then the growth situation and the utilization of mannitol in mutant strains were analyzed.Firstly,the synthesis pathway and the key enzymes of mannitol with glucose as substrate was studied.The recombination strain BL21(DE3)/pET28a-mtlD-mlp was constructed.Then the expression of two genes were analyzed by SDS-PAGE.The results indicated that mtlD expressed highly and the level of mlp expression was very low;the mannitol wasn't detected in fermentation broth with glucose as substrate.Secondly,the synthesis pathway and the key enzymes of mannitol with fructose as substrate were studied.Two recombination strains were constructed,BL21/pET28a-mdh(NADH)and BL21/pET28a-mdh(NADPH)respectively.The activity of NADPH dependent mannitol dehydrogenase(MDH-1)which had been purified was 270 U/mL and the optimum conditions of transforming fructose into mannitol were as follows:300 g/L substrate fructose,pH 5.8,9 mmol/L NADH,and incubation at 40?.Under the optimized conditions,the transformation rate of mannitol reached 97.4%.The activity of NADH dependent mannitol dehydrogenase(MDH-2)which were purified was 173 U/mL and the optimum conditions of transforming fructose into mannitol were as follows:300 g/L substrate fructose,pH 5.3,7.5 mmol/L NADH,and incubation at 30?.Under the optimized conditions,the transformation rate of mannitol reached 92.3%.Then,the decomposition pathway of mannitol in E.coli K-12 were blocked up.Two mutant strains,K-12(?cmtA?cmtB)and K-12(?cmtA?cmtB?mtlA)were obtained by knocking out the gene cmtA,cmtB,mtlA in E.coli PTS system.The growth conditions of original strain and mutant strain were determined.Whereas the growth rate of the mutant strain K-12(?cmtA?cmtB)didn't decrease;the growth rate of the mutant strain K-12(?cmtA?cmtB?mtlA)decreased obviously.The two mutant strains were cultured with mannitol,glucose and the combination as the sole carbon source respectively and determined the growth curves.The growth rate of strain K-12(?cmtA?cmtB)decreased obviously,and the strain K-12(?cmtA?cmtB?mtlA)was almost impossible to grow,which indicated that the mutant strain can't grow with mannitol after knocking out the three genes.Finally,the strain K-12(?cmtA?cmtB?mtlA)/pTrc99a-mdh was constructed.The activity of purified MDH-2 was 258 U/mL,the transformation rate of mannitol reached 3.6%by transforming fructose with cellular fragment.