解除硝酸盐呼吸和氮通量限制对钝齿棒杆菌产精氨酸的影响  

Effect of nitrate respiration and nitrogen flux restriction on arginine production of Corynebacterium crenatum

作　　者：张豪 利霞兰 黄明珠 陈雪岚[1,2,3] ZHANG Hao;LI Xialan;HUANG Mingzhu;CHEN Xuelan(College of Life Science and National R&D Center for Freshwater Fish Processing,Jiangxi Normal University,Nanchang 330027,China;Health College,Jiangxi Normal University,Nanchang 330027,China;College of Sciences,Jiangxi Normal University,Nanchang 330027,China)

机构地区：[1]江西师范大学生命科学学院,国家淡水鱼加工技术研发专业中心,江西南昌330027 [2]江西师范大学健康学院,江西南昌330027 [3]江西师范大学生命科学学院,江西南昌330027

出　　处：《食品与发酵工业》2023年第7期1-8,共8页Food and Fermentation Industries

基　　金：国家自然科学基金项目(31960014);江西省自然科学基金重点项目(20202ACBL205001);江西省教育厅研究生创新基金项目(YC2021-S265)。

摘　　要：精氨酸广泛应用于食品、医药和工业领域中,其生产方式主要是微生物发酵法。钝齿棒状杆菌(Corynebacterium crenatum)是精氨酸生产的重要工程菌株。该文针对课题组前期构建的钝齿棒杆菌(CCM01)进行代谢改造,探究了arnR和cgl 2482的敲除对钝齿棒杆菌产精氨酸的影响。采用无痕敲除方法构建工程菌株并对其进行摇瓶发酵,测定菌体生长量、L-精氨酸产量、葡萄糖消耗量考察菌株产L-精氨酸的影响。硝酸盐能提高钝齿棒杆菌在氧气不足时的生长率,并且arnR的敲除有助于L-精氨酸的生产,arnR敲除菌株CCM02较对照菌株产量提高了8.58%,且当硝酸盐浓度在1.5 mmol/L时对积累L-精氨酸最为有利,产量达到17.09 g/L;cgl 2482的敲除有助于氮源流入精氨酸合成途径,其敲除菌株CCM03精氨酸产量达到了17.88 g,较对照提高了4.9%;最终在CCM03发酵中添加1 g/L的尿素、50 g/L的谷氨酸钠以及1.5 mmol/L的硝酸盐时,L-精氨酸产量达到22.25 g/L,较CCM01提高了46.8%。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.NH 4+is the most preferred nitrogen source for most microorganisms,but high concentrations of NH 4+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 cgl 2482 is to catalyze the decomposition of glutamine into glutamate and NH 4+,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 t

关 键 词：钝齿棒杆菌 arnR 硝酸盐 cgl 2482 氮源 精氨酸生产 

分 类 号：TQ922[轻工技术与工程—发酵工程]