Metabolic Engineering Of Escherichia Coli For D-allose Synthesis And Its Fermentation Optimization

D-allose is a rare hexose with broad market prospects because of its anti-cancer,anti-tumor,anti-inflammatory,anti-oxidation,immunosuppression,cryoprotection,neuroprotection,prevention of hypertension and osteoporosis functions in the fields of medicine,diet and health care.At present,D-allose is generally synthesized by chemical method or enzymatic method,but there are bottlenecks in both methods,which astrict the large-scale production of D-allose.In this study,the rationally designed cell synthesis plant and fermentation conditions of D-allose are optimized in order to achieve efficient synthesis of D-allose.Escherichia coli JM109(DE3),D-glucose and glycerol serve as the chassis host,substrate,and metabolic carbon source,by coexpressing exogenous genes gi,dpe,rpiB,and overexpressing endogenous gene galP,the pathway to synthesis D-allose in cell was successfully constructed.Knock out fruA and ptsG with recombination to relieve the carbon catabolite repression(CCR)effect of D-glucose on glycerol transport.Further,by knockouting of pfkA,glk,mak and pfkB,the metabolic flow of D-glucose and D-fructose into the glycolysis pathway is blocked,and the substrate conversion of D-allose is improved.Through response surface analysis and optimization of key fermentation parameters(temperature and IPTG concentration),the optimal fermentation temperature and IPTG concentration were determined as 30? and 0.2mM,respectively.The output of D-allose reached 19.01mg/L by shaking flask fermentation on M9 mixed sugar medium(5g/L D-glucose and 7g/L glycerol)under the optimal fermentation conditions.