ZHANG Hao, LI Xialan, HUANG Mingzhu, CHEN Xuelan
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.
