| The development of amino acid production process has undergone thermal acid hydrolysis,chemical synthesis,enzyme catalysis and microbial fermentation.With the increase of population and the further exploitation of various amino acid functions,its market demands have increased steadily by 5-7% per year.L-arginine,as a semi-essential amino acid,participates in the urea cycle and nitrogen metabolism in the human body,and helps ammonia to be discharged.It also participates in NO synthesis and indirectly affects the immune,nervous and cardiovascular systems of living body;as one of the important components of protamine,arginine affects human reproduction.Therefore,it is widely used as an additive for foods,medicines,and et al.Currently,arginine in China mainly relies on foreign imports,and is obtained by acid hydrolysis from domestic pig hair and animal skin as raw materials.However,regardless of environmental pollution or economic saving,the use of engineering bacteria fermentation is the most right way.Corynebacterium crenatum is gram-positive bacterium isolated from soil by domestic researchers.Because its genome is highly similar to C.glutamicum,and has the same advantages as C.glutamicum,the strain is used widely to produce various amino acids,including arginine.In this paper,C.crenatum MT argB M4?TSCP?pta?proB?cg12310?Ncgl1221 and C.crenatum MT argB M4?TSCP?pta?proB?cg12310?Ncgl1221?cobB::cg3035 were used as the starting strains.The high-producing arginine strain was obtained using homologous recombination technology to intervene transcriptional regulators,byproduct tributaries,NADPH reductive force pools,and arginine metabolic pathway carbon fluxes.Firstly,the original promoter of farR was replaced by the lacI promoter from Escherichia coli in C.crenatum MT argB M4?TSCP?pta?proB?cg12310?Ncgl1221.The results of fluorescent quantitative PCR showed that the expression levels of arginine synthesis genes gdh and argB were down-regulated,and the expression levels of zwf and gnd involved in NADPH synthesis were down-regulated,while the expression of the positive correlation gene odhA with ODHC activity was up-regulated,and the expression of negative correlation gene odhI with ODHC activity was down-regulated.The results of shake flask fermentation also showed that the cell growth and glucose consumption were no difference,but arginine production decreased by about 15%,which indicated that FarR is not the negative regulator in arginine synthetic network.Secondly,the key gene speE of putrescine synthesis was knocked out by homologous recombination in C.crenatum MT argB M4?TSCP?pta?proB?cg12310?Ncgl1221?cobB::cg3035,which cut off the byproduct branch of ornithine catabolism.The yield of arginine increased with the fermentation time,and the yield was impoved by 25% at 120 hours.Finally,the gapC from Clostridium acetobutylicum was heterologously expressed in C.crenatum MT argB M4?TSCP?pta?proB?cg12310?Ncgl1221?cobB::cg3035,which can catalyze the glycerol 3-phosphate and increase NADPH accumulation.The results of shake flask fermentation showed that the sugar consumption was reduced but the growth of the bacteria increased,and the arginine production increased by 15%.In addition,NADH-dependent glutamate dehydrogenase gene rocG from Bacillus subtilis was heterologous expressed at the position of the strain ?proB,and the arginine production increased by 21.3%compared with the control strain.In conclusion,the construction methods of high-producing arginine strain were explored in C.crenatum by disruption of speE for increasing synthetic pathway carbon flux,heterologous expression of rocG and gapC for increasing NADPH pool,as well as weakening farR. |
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