Effects Of Nitrate And Mutagenesis Breeding On Arginine Production Of Corynebacterium Crenatum

L-arginine is a very important non-essential amino acid for human body,which can be used as a precursor of NO production and participate in the urea cycle.At present,the main production method of L-arginine is microbial fermentation.How to further improve L-arginine production has become the focus of the industry.In this paper,the growth and arginine production of C.crenatum were firstly studied by adding nitrate in the high-density fermentation in the later fermentation period.The result showed that C.crenatum can use nitrate for anaerobic growth.Then,CCM02 was constructed by deletion of inhibitory factor arnR of operon nar KGHJI,and further optimized the fermentation process.In order to breed L-arginine high-yielding strains,two strains CCM03 and CCM04 were successfully bred by arginine analogue mutagenesis and UV mutagenesis.Then,the mutant with the highest yield was used as the starting strain to optimize the addition time and amount of nitrate.The fermentation results of shaking flask showed as follows:(1)When 1.5mmol/L nitrate was added in the fermentation of CCM01,L-Arg was the highest,reaching 13.058g/L,which was 11.3% higher than that of the control group.Adding 3mmol/L nitrate inhibited the growth of CCM01.(2)L-Arg production of arnR-deleted strain CCM02 reached 13.058g/L by adding 3 mmol/L nitrate,which was 12.39% higher than that of the control group.It can be seen that arnR knockout can enable C.crenatum to grow in higher concentration of nitrate.(3)Adding nitrate during the fermentation of mutant strains CCM03 and CCM04,the results showed that when adding 3mmol/L nitrate in CCM03,the final L-Arg produced by fermentation reached 16.617g/L,and the yield of L-Arginine of CCM03 strain increased by 13.2% compared with that of CCM03 strain without adding nitrate.L-Arg yield of CCM04 strain was 15.934g/L when 3mmol nitrate was added,which was 11.37% higher than that of L-Arginine of CCM04 strain without nitrate.(4)The fermentation optimization results of mutant CCM03 showed that when10mmol/L nitrate was added at 72 hours,L-Arg arginine yield reached the maximum of 22.36g/L,90.6% higher than CCM01.