L-酪氨酸高产菌株的构建及发酵培养基优化  

Construction of high-yield L-tyrosine strain and fermentation medium optimization

作　　者：杜丽红 吕思琪 战俊杰 马志鹏 马丽媛[1] 马雪[1] 范恒军 孟天星 DU Lihong;LV Siqi;ZHAN Junjie;MA Zhipeng;MA Liyuan;MA Xue;FAN Hengjun;MENG Tianxing(College of Food and Pharmaceutical Engineering,Suihua University,Suihua 152000,China;Xiangyu Jingu Biochemical Technology Co.,Ltd.,Suihua 152000,China)

机构地区：[1]绥化学院食品与制药工程学院,黑龙江绥化152000 [2]象屿金谷生化科技有限公司,黑龙江绥化152000

出　　处：《中国酿造》2023年第11期202-208,共7页China Brewing

基　　金：黑龙江省教育厅基本科研业务费项目(YWK10236210232);黑龙江省大学生创新创业训练计划项目(S202210236004);黑龙江省自然科学基金(JJ2022LH1055);黑龙江省青年科技人才托举工程项目(2022QNTJ08)。

摘　　要：该研究以L-酪氨酸合成的两个关键基因aroG和tyrR为研究靶点,在大肠杆菌(Escherichia coli)TRP1中上调表达解除反馈抑制的基因aroG,强化莽草酸途径,同时敲除基因tyrR,解除TyrR蛋白对aroG、tyrB、aroF、tyrA、aroL、aroP、tyrP多个基因的转录抑制,构建高产L-酪氨酸的重组工程菌株,并以L-酪氨酸产量为评价指标,采用单因素及正交试验对重组工程菌株的发酵培养基进行优化。结果表明,获得一株高产L-酪氨酸的重组菌株TY03,L-酪氨酸产量为(17.4±0.15)g/L,是出发菌株大肠杆菌TRP1的86倍,其产L-酪氨酸的最优发酵培养基为:酵母粉8 g/L、MgSO_(4)·7H2O 4 g/L、FeSO_(4)·7H2O 40 mg/L、MnSO_(4)·H2O 40 mg/L、VB15 mg/L、VH 8 mg/L、(NH_(4))_(2)SO_(4)5 g/L、KH2PO_(4)3 g/L,在此条件下,L-酪氨酸产量为(20.9±0.19)g/L,比优化前提高了20%。该研究优化重组菌株TY03的培养基组成,同时对于大肠杆菌高效积累L-酪氨酸的定向改造提供了一种新思路。Using the two key genes aroG and tyrR for L-tyrosine synthesis as research targets,the remove feedback inhibition gene aroG was up-regulate expressed,the shikimic acid pathway was strengthened,gene tyrR was knocked out at the same time,the transcriptional inhibition of TyrR protein on aroG,tyrB,aroF,tyrA,aroL,aroP,and tyrP genes was removed,and the high-yield L-tyrosine recombinant engineered strain was constructed.Using L-tyrosine yield as evaluation index,the fermentation medium of recombinant engineered strain was optimized by single factor and orthogonal experiments.The results showed that a recombinant strain TY03 with high-yield L-tyrosine was obtained,and the L-tyrosine yield was(17.4±0.15)g/L,which was 86 times that of the starting strain Escherichia coli TRP1.The optimal fermentation medium compositions for L-tyrosine production were as follows:yeast powder 8 g/L,MgSO_(4)·7H_(2)O 4 g/L,FeSO_(4)·7H_(2)O 40 mg/L,MnSO_(4)·H2O 40 mg/L,VB15 mg/L,VH 8 mg/L,(NH_(4))2SO_(4)5 g/L,and KH2PO_(4)3 g/L.Under these conditions,L-tyrosine yield was(20.9±0.19)g/L,which increased 20%than before optimization.The medium compositions of reco-mbinant strain TY03 were optimized,and a new idea for the targeted modification of L-tyrosine accumulation in E.coli.

关 键 词：大肠杆菌 L-酪氨酸 代谢工程 发酵培养基 正交试验 

分 类 号：TQ922[轻工技术与工程—发酵工程]