谷氨酸棒状杆菌合成5-氨基乙酰丙酸的途径构建与发酵优化  

Construction of 5-aminolevulinic Acid Synthesis Pathway and Optimization of Fermentation by Corynebacterium glutamicum

作　　者：饶德明 张良程 陈久洲[3,2] 孙德虎[1,2] 孙村民[3,2] 郑小梅[3,2] 郑平[3,2] 刁爱坡[1] RAO De-ming ZHANG Liang-cheng CHEN Jiu-zhou SUN De-hu SUN Cun-min ZHENG Xiao-mei ZHENG Ping DIAO Ai-po(School of Biological Engineering, Tianjin University of Scienee and Technology, Tianjin 300457 Key Laboratory of Systems Microbial Biotechnology , Chinese Academy of Sciences, Tianjin 300308 Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院天津工业生物技术研究所,天津300308 [3]中国科学院系统微生物工程重点实验室,天津300308

出　　处：《生物技术通报》2017年第1期148-156,共9页Biotechnology Bulletin

基　　金：天津市科技支撑计划重点项目(13ZCZDSY04900)

摘　　要：5-氨基乙酰丙酸(5-amino levulinic acid,ALA)是生物体内天然存在的一种非蛋白质氨基酸,在农业和医药等领域应用广泛。为了在谷氨酸棒状杆菌中建立ALA碳四合成途径并优化其发酵体系,首先在谷氨酸棒状杆菌中过表达沼泽红假单胞菌来源的ALA合成酶(ALAS),建立高效的ALA碳四合成途径,然后从不同发酵培养基的比较、诱导剂和底物甘氨酸的浓度以及初始接种量等不同方面对ALA摇瓶发酵工艺进行了优化。结果显示,过表达Hem A的13032/p ZWA1菌株ALA产量达到1.41 g/L,是对照菌株的67.14倍。ALA的最优摇瓶发酵条件为以酵母粉为氮源的M9培养基,采用5%的接种量0.1 mmol/L IPTG进行Hem A的诱导表达,体系中甘氨酸的浓度要控制在4 g/L,摇瓶中ALA产量可达到3.28 g/L,比优化前提高了132.62%。采用发酵优化的条件,在5 L发酵罐的放大发酵中ALA产量可达10.08 g/L,这是现有报道中谷氨酸棒状杆菌一步发酵合成ALA的最高产量。5-aminolevulinic acid（ALA）is a natural non-protein amino acid and has been widely applied in agriculture and medicine.This study aimed to construct the systhesis pathway of C4 ALA in Corynebacterium glutamicum and optimize its fermentation process. First,efficient C4 ALA synthesis pathway was established in C. glutamicum by over-expressing the ALA synthase（ALAS）from Rhodopseudomonas palustris. Then,the ALA fermentation process with flask was optimized from four factors ：fermentation medium,concentration of inducer,substrate concentration,and initial inoculum dosage. As results,the ALA yield of the strain 13032/p ZWA1 over-expressing Hem A was 1.41g/L,up to 67.14 folds compared with the control strain. The optimal ALA fermentation condition was M9 media using yeast extract as nitrogensource with 5% inoculum size,0.1 mmol/L IPTG to induce the experession of Hem A,and the concentration of glycine must be at 4 g/L. Afteroptimization,ALA yield reached 3.28 g/L in a shaking flask and increased 132.62% than before. In conclusion,under the optimal fermentationcondition,the ALA yield in a 5 L bioreactor fermentation was 10.08 g/L,which was the highest ALA yield by one-step fermentation of C. glutamicum reported so far.

关 键 词：5-氨基乙酰丙酸 谷氨酸棒状杆菌 5-氨基乙酰丙酸合成酶 沼泽红假单胞菌 发酵优化 

分 类 号：TQ922[轻工技术与工程—发酵工程]