限氧发酵生产缬氨酸工程菌株及发酵过程优化  

Optimization of engineered strain and fermentation process for valine production via oxygen-limited fermentation

作　　者：王加初 伍法清 吴鹤云 谢希贤[1] WANG Jiachu;WU Faqing;WU Heyun;XIE Xixian(College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China;College of Food Science and Engineering,Tianjin University of Science and Technology,Tianjin 300457,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]天津科技大学食品科学与工程学院,天津300457

出　　处：《食品与发酵工业》2023年第1期33-39,46,共8页Food and Fermentation Industries

基　　金：国家重点研发计划项目(2018YFA0900300)。

摘　　要：VHY18是之前构建的可在限氧条件下高产缬氨酸的菌株,但在发酵过程中会大量积累琥珀酸和乙酸,生长活力也会显著衰退,限制其发酵性能。为此,该研究首先敲除了VHY18中丙酮酸氧化酶和乙酸激酶编码基因poxB和ackA,构建了VHY19,使乙酸浓度降低了43.7%。接着敲除了VHY19的苹果酸脱氢酶编码基因mdh,构建了VHY20,使琥珀酸浓度降低了50.8%。最后优化了VHY20的双阶段发酵工艺,主要摸索了限氧发酵阶段的供氧条件、2个阶段的转换时机及葡萄糖流加速率。优化后的VHY20双阶段发酵工艺为,发酵开始时先进行好氧发酵,14 h后将好氧发酵转换至限氧发酵,并将供氧条件设置为搅拌转速400 r/min、风量2 L/min,补糖速率控制在25 g/h。菌株和发酵过程优化后,缬氨酸产量为90.4 g/L,转化率为0.53 g/g葡萄糖,分别较优化前提高了7.8%和30.5%;乙酸和琥珀酸的积累量分别为2.7和1.2 g/L,较优化前降低了76.9%和95.4%。The VHY18 strain capable of high-yield valine production under oxygen-limited conditions was previously constructed.However,a large amount of succinate and acetate were accumulated during the fermentation process and the strain growth declined significantly,limiting the fermentation performance.To this end,the genes encoding the pyruvate oxidase and acetate kinase,poxB and ackA,were firstly knocked out from VHY18 in this study,generating VHY19,and enabled acetate concentration to decrease by 43.7%.Then the mdh gene encoding the malate dehydrogenase of WHY19 was knocked out to construct VHY20,contribute to decrease of acetate concentration by 43.7%.Finally,the two-stage fermentation process of VHY20 was optimized,mainly exploring the oxygen supply conditions in the oxygen-limited fermentation stage,the switch timing between the two stages,and the supplementation rate of glucose.The optimized two-stage fermentation process of VHY20 was as follows:aerobic fermentation was carried out at the beginning of the fermentation,and after 14 h,the aerobic fermentation was switched to oxygen-limited fermentation,and the oxygen supply conditions were set as stir speed of 400 r/min,air flow of 2 L/min and the glucose supplementation rate was controlled at 25 g/h.Through the optimization of the strain and fermentation process,the valine titer was up to 90.4 g/L with the yield of 0.53 g/g glucose,increasing by 7.8%and 30.5%respectively and the succinate and acetate titers were down to 2.7 and 1.2 g/L,decreasing by 76.9%and 95.4%respectively compared with results before optimization.

关 键 词：缬氨酸 代谢工程 发酵优化 大肠杆菌 限氧发酵 

分 类 号：TQ922[轻工技术与工程—发酵工程]