Cloning, Expression Of The Key Enzymes Of The EMP Pathway In Corynebacterium Crenatum And Their Effects On L-arginine Synthesis

L-arginine has a very wide range of applications in medicine, food and other fieldsbecause of its physiological and pharmacological effects. Corynebacterium crenatum SYPA5-5which has a high L-arginine yield was obtained by multi-level mutagenesis. EMP pathwayis the common pathway for glucose catabolism in vivo. Hexokinase, phosphofructokinaseand pyruvate kinase catalyze irreversible reactions and play an important role in regulation ofEMP pathway.The genes encoding glucokinase, phosphofructokinase and pyruvate kinase in EMPpathway from C. crenatum were cloned and expressed in E. coli BL21. The correspondingenzymes GLK, PFK and PYK were purified by Ni-NTA and the enzymatic characterizationswere detected. The specific enzyme activity of the purified GLK was58.91U mg-1and therecovery ratio was81.73%. Then the enzyme characterization was studied. The resultsshowed that the optimum pH was7.5, the optimum reaction temperature was30°C, and theKmvalue for glucose was0.159mmol L-1. The specific enzyme activity of the purified PFKwas109.96U mg-1and the recovery ratio was84.70%. Then the enzyme characterization wasstudied. The results showed that the optimum pH was7.0, the optimum reaction temperaturewas25°C, and the Kmvalue for fructose-6-phosphate was0.076mmol L-1. The specificenzyme activity of the purified PYK was188.64U mg-1and the recovery ratio was78.16%.Then the enzyme characterization was studied. The results showed that the optimum pH was7.5, the optimum reaction temperature was45°C, and the Kmvalue for phosphoenolpyruvatewas0.120mmol L-1.The genes encoding the GLK, PFK and PYK were over-expressed in C. crenatum SYPA5-5. The flask fermentation of recombinant strains exhibited that over-expression of PFK, theL-arginine yield reached34.7g·L-1, which is increased15.3%than the strain SYPA5-5.When PYK was over-expressed in C. crenatum, the L-arginine yield was35.0g·L-1, which isincreased16.2%in the flask fermentation. In the5L jar, the recombinant strain whichover-expressed of PFK showed that the L-arginine yield reached42.5g·L-1and the maximumproduction rate of L-arginine was0.54g·L-1·h-1, which are increased17.7%and17.4%thanthe strain SYPA5-5, respectively. While, the over-expressed of PYK showed that theL-arginine yield reached43.3g·L-1and the maximum production rate of L-arginine was0.55g·L-1·h-1, which are increased19.9%and19.6%than the strain SYPA5-5, respectively.Then the genes encoding PFK and PYK were co-expressed in C. crenatum SYPA5-5.The results exhibited that the L-arginine production could reach39.6g·L-1, about24.9%higher compared with the strain SYPA5-5. In the5L jar, the L-arginine production reached48.7g·L-1and the maximum production rate of L-arginine was0.63g·L-1·h-1, which are increased27.5%and28.6%than the initial strain, respectively. The enzymatic properties ofGLK, PFK and PYK were studied and all of the results provided reliable theory basis forculture medium optimization and fermentation process improvement. Our strategy not onlysuccessfully improved the L-arginine production by co-expression of PFK and PYK in C.crenatum SYPA5-5but also provided practice basis for reveal the effect of central carbonmetabolism on L-arginine synthesis.