Arginine is widely used in food, medicine and industrial fields, and its production method is mainly microbial fermentation. Corynebacterium crenatum is an important engineered strain for arginine production. As a facultative aerobic bacterium, it is crucial to ensure a certain level of oxygen supply during fermentation. However, any aerobic bacteria fermentation industry in the late fermentation, and the dissolution of oxygen absorption will be interfered with high density of bacteria. In this case, even improvement of bioreactor ventilation and stirring speed can also be difficult to meet the oxygen demand of arginine production. In the late of the aerobic fermentation, accompanied by a lot of anaerobic fermentation process, intracellular metabolism is directed towards the synthesis of unwanted compounds such as lactic acid and alcohol, which severely inhibits the accumulation of target metabolites. Many microorganisms have nitrate respiration systems, using nitrate as an electron acceptor to generate energy for their growth and metabolism under hypoxic conditions. The results showed that the transcription product of arnR was a negative regulator of nitrate reductase. Thus, decrease of its expression could relieve the restriction of nitrate respiration in C. crenatum under aerobic conditions, and promote the accumulation of arginine. In addition, as the amino acid with the highest nitrogen content, the synthesis of L-arginine was closely related to the absorption and utilization efficiency of nitrogen sources. NH4+ is the most preferred nitrogen source for most microorganisms, but high concentrations of NH4+ have toxic effects on the growth of bacteria. Therefore, promoting the assimilation process of ammonia is necessary for the synthesis of arginine. The main function of glutaminase encoded by cgl2482 is to catalyze the decomposition of glutamine into glutamate and NH4+, and its activity is not conducive to the assimilation of ammonia and the synthesis of intracellular nitrogen sources flowing into arginine. In this paper, the metabolic transformation of C. crenatum (CCM01) previously constructed by the research group was carried out, and the effect of knockout of arnR and cgl2482 on the production of arginine by C. crenatum was explored. The engineered strains were constructed by the traceless knockout method and subjected to shake flask fermentation. The bacterial growth, L-arginine production and glucose consumption were determined to investigate the effect of the strains on L-Arg production. Nitrate could increase the growth rate of C. crenatum on lack of oxygen, and the arnR knockout contributed to the accumulation of L-arginine. The arnR knockout strain CCM02 increased the yield by 8.58% compared with the control strain, and when the nitrate concentration of 1.5 mmol/L was the most favorable for the accumulation of L-arginine, and the yield reached 17.09 g/L; the knockout of cgl2482 helped the nitrogen source to flow into the arginine synthesis pathway, and its knockout strain CCM03 arginine The yield reached 17.88 g, which was 4.9% higher than the control. Finally, when 1 g/L urea, 50 g/L sodium glutamate and 1.5 mmol/L nitrate were added to the CCM03 fermentation, the L-arginine yield reached 22.25 g/L, 46.8% higher than CCM01. The research results can be used as a new strategy to obtain arginine high-producing strains.
[1] JIANG Y, SHENG Q, WU X Y, et al.L-arginine production in Corynebacterium glutamicum:Manipulation and optimization of the metabolic process[J].Critical Reviews in Biotechnology, 2021, 41(2):172-185.
[2] HERNÁNDEZ V M, ARTEAGA A, DUNN M F.Diversity, properties and functions of bacterial arginases[J].FEMS Microbiology Reviews, 2021, 45(6):fuab034.
[3] 赵越, 朱凌峰, 詹丽, 等.黄素还原酶Frd181和Frd188对钝齿棒杆菌产精氨酸的影响[J].生物技术, 2020, 30(1):69-74.
ZHAO Y, ZHU L F, ZHAN L, et al.Comparsion of the effect of flavin reductase Frd181 and Frd188 on arginine production of Corynebacterium crenatum[J].Biotechnology, 2020, 30(1):69-74.
[4] GHOLAMI M, BOUGHTON B A, FAKHARI A R, et al.Metabolomic study reveals a selective accumulation of L-arginine in the D-ornithine treated tobacco cell suspension culture[J].Process Biochemistry, 2014, 49(1):140-147.
[5] HU J L, LEI P, MOHSIN A, et al.Mixomics analysis of Bacillus subtilis:Effect of oxygen availability on riboflavin production[J].Microbial Cell Factories, 2017, 16(1):150.
[6] KABORÉ A K, OLMOS E, FICK M, et al.Aerobiosis-anaerobiosis transition has a significant impact on organic acid production by Corynebacterium glutamicum[J].Process Biochemistry, 2017, 52:10-21.
[7] HASEGAWA S, TANAKA Y, SUDA M, et al.Enhanced glucose consumption and organic acid production by engineered Corynebacterium glutamicum based on analysis of a pfkB1 deletion mutant[J].Applied and Environmental Microbiology, 2017, 83(3):e02638-16.
[8] OKINO S, SUDA M, FUJIKURA K, et al.Production of D-lactic acid by Corynebacterium glutamicum under oxygen deprivation[J].Applied Microbiology and Biotechnology, 2008, 78(3):449-454.
[9] YASUDA K, JOJIMA T, SUDA M, et al.Analyses of the acetate-producing pathways in Corynebacterium glutamicum under oxygen-deprived conditions[J].Applied Microbiology and Biotechnology, 2007, 77(4):853-860.
[10] EMERSON D F, WOOLSTON B M, LIU N, et al.Enhancing hydrogen-dependent growth of and carbon dioxide fixation by Clostridium ljungdahlii through nitrate supplementation[J].Biotechnology and Bioengineering, 2019, 116(2):294-306.
[11] TAKENO S, OHNISHI J, KOMATSU T, et al.Anaerobic growth and potential for amino acid production by nitrate respiration in Corynebacterium glutamicum[J].Applied Microbiology and Biotechnology, 2007, 75(5):1 173-1 182.
[12] FAN L W, WANG Y, QIAN J, et al.Transcriptome analysis reveals the roles of nitrogen metabolism and sedoheptulose bisphosphatase pathway in methanol-dependent growth of Corynebacterium glutamicum[J].Microbial Biotechnology, 2021, 14(4):1 797-1 808.
[13] UNDEN G, KLEIN R.Sensing of O2 and nitrate by bacteria:Alternative strategies for transcriptional regulation of nitrate respiration by O2 and nitrate[J].Environmental Microbiology, 2021, 23(1):5-14.
[14] NISHIMURA T, TERAMOTO H, INUI M, et al.Corynebacterium glutamicum ArnR controls expression of nitrate reductase operon narKGHJI and nitric oxide (NO)-detoxifying enzyme gene hmp in an NO-responsive manner[J].Journal of Bacteriology, 2014, 196(1):60-69.
[15] HUANG M Z, ZHU L F, FENG L, et al.Reforming nitrate metabolism for enhancing L-arginine production in Corynebacterium crenatum under oxygen limitation[J].Frontiers in Microbiology, 2022, 13:834311.
[16] PARK S H, KIM H U, KIM T Y, et al.Metabolic engineering of Corynebacterium glutamicum for L-arginine production[J].Nature Communications, 2014, 5(1):1-9.
[17] YANG X Y, XU M Y, ZOU R S, et al.Microbial protein production from CO2, H2, and recycled nitrogen:Focusing on ammonia toxicity and nitrogen sources[J].Journal of Cleaner Production, 2021, 291:125921.
[18] BURKOVSKI A.Ammonium assimilation and nitrogen control in Corynebacterium glutamicum and its relatives:An example for new regulatory mechanisms in actinomycetes[J].FEMS Microbiology Reviews, 2003, 27(5):617-628.
[19] GUO J, MAN Z W, RAO Z M, et al.Improvement of the ammonia assimilation for enhancing L-arginine production of Corynebacterium crenatum[J].Journal of Industrial Microbiology & Biotechnology, 2017, 44(3):443-451.
[20] HUANG M Z, ZHAO Y, LI R, et al.Improvement of L-arginine production by in silico genome-scale metabolic network model guided genetic engineering[J].3 Biotech, 2020, 10(3):126.
[21] 钱和, 郝刚.L-精氨酸产生菌诱变育种的研究[J].微生物学通报, 2005, 32(3):46-50.
QIAN H, HAO G.Study on the breeding of L-arginine-producing strain[J].Microbiology, 2005, 32(3):46-50.
[22] ZHANG B, WAN F, QIU Y L, et al.Increased L-arginine production by site-directed mutagenesis of N-acetyl-L-glutamate kinase and proB gene deletion in Corynebacterium crenatum[J].Biomedical and Environmental Sciences, 2015, 28(12):864-874.
[23] 赵凯,许鹏举,谷广烨.3,5-二硝基水杨酸比色法测定还原糖含量的研究[J].食品科学, 2008, 29(8):534-536.
ZHAO K, XU P J, GU G Y.Study on determination of reducing sugar content using 3,5-Dinitrosalicylic acid method[J].Food Science, 2008, 29(8):534-536.
[24] 万方, 张斌, 陈民良, 等.proC及putP基因的敲除对钝齿棒杆菌产L-精氨酸生理代谢的影响[J].中国生物工程杂志, 2015, 35(8):51-58.
WAN F, ZHANG B, CHEN M L, et al.Effects of pro C and put P deletion on physiological metabolism of L-arginine producing strain Corynebacterium crenatum[J].China Biotechnology, 2015, 35(8):51-58.
[25] PÉREZ-ÁLVAREZ E P, GARDE-CERDÁN T, GARCÍA-ESCUDERO E, et al.Effect of two doses of urea foliar application on leaves and grape nitrogen composition during two vintages[J].Journal of the Science of Food and Agriculture, 2017, 97(8):2 524-2 532.
[26] LYU Q L, HU M K, TIAN L Z, et al.Enhancing L-glutamine production in Corynebacterium glutamicum by rational metabolic engineering combined with a two-stage pH control strategy[J].Bioresource Technology, 2021, 341:125799.
[27] MUGITA Y, NAKAGAMI G, MINEMATSU T, et al.Combination of urease inhibitor and antiseptic inhibits urea decomposition-induced ammonia production by Proteus mirabilis[J].International Wound Journal, 2020, 17(6):1 558-1 565.
