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通过大肠杆菌中的温度调节开关高效生产L-丙氨酸

Efficient L-Alanine Production by a Thermo-Regulated Switch in Escherichia coli.

作者信息

Zhou Li, Deng Can, Cui Wen-Jing, Liu Zhong-Mei, Zhou Zhe-Min

机构信息

The Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, People's Republic of China.

Center for Bioresource & Bioenergy, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, People's Republic of China.

出版信息

Appl Biochem Biotechnol. 2016 Jan;178(2):324-37. doi: 10.1007/s12010-015-1874-x. Epub 2015 Oct 9.

Abstract

L-Alanine has important applications in food, pharmaceutical and veterinary and is used as a substrate for production of engineered thermoplastics. Microbial fermentation could reduce the production cost and promote the application of L-alanine. However, the presence of L-alanine significantly inhibit cell growth rate and cause a decrease in the ultimate L-alanine productivity. For efficient L-alanine production, a thermo-regulated genetic switch was designed to dynamically control the expression of L-alanine dehydrogenase (alaD) from Geobacillus stearothermophilus on the Escherichia coli B0016-060BC chromosome. The optimal cultivation conditions for the genetically switched alanine production using B0016-060BC were the following: an aerobic growth phase at 33 °C with a 1-h thermo-induction at 42 °C followed by an oxygen-limited phase at 42 °C. In a bioreactor experiment using the scaled-up conditions optimized in a shake flask, B0016-060BC accumulated 50.3 g biomass/100 g glucose during the aerobic growth phase and 96 g alanine/100 g glucose during the oxygen-limited phase, respectively. The L-alanine titer reached 120.8 g/l with higher overall and oxygen-limited volumetric productivities of 3.09 and 4.18 g/l h, respectively, using glucose as the sole carbon source. Efficient cell growth and L-alanine production were reached separately, by switching cultivation temperature. The results revealed the application of a thermo-regulated strategy for heterologous metabolic production and pointed to strategies for improving L-alanine production.

摘要

L-丙氨酸在食品、制药和兽医领域有重要应用,还被用作生产工程热塑性塑料的底物。微生物发酵可以降低生产成本并促进L-丙氨酸的应用。然而,L-丙氨酸的存在会显著抑制细胞生长速率,并导致最终L-丙氨酸生产率下降。为了高效生产L-丙氨酸,设计了一种温度调节的基因开关,以动态控制嗜热栖热放线菌的L-丙氨酸脱氢酶(alaD)在大肠杆菌B001–060BC染色体上的表达。使用B001–060BC进行基因开关丙氨酸生产的最佳培养条件如下:在33°C进行好氧生长阶段,在42°C进行1小时的热诱导,然后在42°C进行限氧阶段。在使用摇瓶中优化的放大条件进行的生物反应器实验中,B001–060BC在好氧生长阶段分别积累了50.3 g生物量/100 g葡萄糖,在限氧阶段积累了96 g丙氨酸/100 g葡萄糖。以葡萄糖为唯一碳源时,L-丙氨酸滴度达到120.8 g/L,总容积生产率和限氧容积生产率分别更高,为3.09和4.18 g/L·h。通过切换培养温度,分别实现了高效的细胞生长和L-丙氨酸生产。结果揭示了温度调节策略在异源代谢生产中的应用,并指出了提高L-丙氨酸生产的策略。

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