谷氨酸棒状杆菌合成L-高丝氨酸的代谢改造与发酵条件探究  

Metabolic Transformation and Fermentation Condition of L-homoserine Synthesis by Corynebacterium glutamicum

作　　者：郭秋爽 包倩倩 许银彪 蔡帅 孙杨 李华[1,2] 刘宇鹏 GUO Qiushuang;BAO Qianqian;XU Yinbiao;CAI Shuai;SUN Yang;LI Hua;LIU Yupeng(Institute of Microbial Engineering,College of Life Sciences,Henan University,Kaifeng 475004,China;Henan Applied Microbial Engineering Research Center,Kaifeng 475004,China)

机构地区：[1]河南大学生命科学学院微生物工程研究所,河南开封475004 [2]河南省应用微生物工程研究中心,河南开封475004

出　　处：《食品工业科技》2023年第3期133-140,共8页Science and Technology of Food Industry

基　　金：河南省自然科学基金资助项目(202300410055);河南省重点研发与推广专项(No.192102110179)。

摘　　要：目的:本研究以谷氨酸棒状杆菌ATCC 13032为底盘细胞,构建1株L-高丝氨酸合成菌株并分析溶氧环境对其产物合成的影响。方法:首先通过外源添加0~40 g/L的L-高丝氨酸分析谷氨酸棒状杆菌的产物耐受性;随后,通过基因thrB敲除阻断L-高丝氨酸的降解途径,获得谷氨酸棒状杆菌重组菌H1;在此基础上利用挡板摇瓶进行细胞培养以增强发酵过程中氧气供给能力。结果:与大肠杆菌相比,谷氨酸棒状杆菌对L-高丝氨酸具有更强耐受性。研究中通过敲除基因thrB构建了L-苏氨酸缺陷型谷氨酸棒状杆菌重组菌H1,发现基础培养基中加入0.5 g/L的L-苏氨酸后,该重组菌生长恢复正常水平。挡板摇瓶条件下重组菌H1的L-高丝氨酸产量增加至836.7 mg/L,较普通摇瓶产量44.6 mg/L提高了17.76倍。结论:通过阻断L-苏氨酸的合成,成功构建L-高丝氨酸合成菌株谷氨酸棒状杆菌H1,并且发现利用挡板摇瓶增强发酵过程中供氧能力是促进谷氨酸棒状杆菌高效合成L-高丝氨酸的有效手段,为后续提高L-高丝氨酸发酵产量提供了参考。Objective:In this study,Corynebacterium glutamicum ATCC 13032 was used as the chassis cell for synthesizing L-homoserine and analyzing the effect of dissolved oxygen on product synthesis.Methods:First,the product tolerance of C.glutamicum was analyzed by exogenously adding 0~40 g/L L-homoserine.Second,the degradation pathway of L-homoserine was blocked by gene thrB knockout,namely C.glutamicum recombinant strain H1.On this basis,the shake flask with baffles was used for cell culture to enhance oxygen supply capacity in the fermentation process.Results:Compared with Escherichia coli,C.glutamicum had a stronger tolerance to L-homoserine.In the study,C.glutamicum recombinant strain H1 was constructed by deleting the gene thrB.It was found that the growth of recombinant strain H1 returned to normal after adding 0.5 g/L L-threonine in the basal medium.The L-homoserine production of recombinant strain H1 increased to 836.7 mg/L using shake flask with baffles,which was 17.76 times higher than that using ordinary shake flask,which was 44.6 mg/L.Conclusion:C.glutamicum recombinant strain H1 was successfully constructed for producing L-homoserine via blocking the synthesis of L-threonine.It was found that the using of shake flask with baffles to enhance the oxygen supply capacity during fermentation was an effective means to promote the production of L-homoserine by C.glutamicum.This study provides a reference for improving L-homoserine production subsequently.

关 键 词：L-高丝氨酸 谷氨酸棒状杆菌重组菌 强耐受性 溶氧 

分 类 号：TS201.3[轻工技术与工程—食品科学]