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

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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.

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Acknowledgments

This work was partly funded by the National Natural Science Foundation of China (31300087, 31400058), the Natural Science Foundation of Jiangsu (BK20130131, BK20130139 and BK20140151), the National High Technology Research and Development Program of China (863 Program, 2014AA021304), the High Foreign Experts Project (GDW20123200114), the Priority Academic Program Development of Jiangsu Higher-Education Institutions, the 111 Project (111-2-06), the Jiangsu Province “Collaborative Innovation Center for Advanced Industrial Fermentation” Industry Development Program, the Public Project for Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University (KLIB-KF201306) and the Fundamental Research Funds for the Central Universities (JUSRP51411B, JUSRP51504).

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Authors and Affiliations

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

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

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

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

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  1. Li Zhou
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  2. Can Deng
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  3. Wen-Jing Cui
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Correspondence to Zhe-Min Zhou.

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Zhou, L., Deng, C., Cui, WJ. et al. Efficient L-Alanine Production by a Thermo-Regulated Switch in Escherichia coli . Appl Biochem Biotechnol 178, 324–337 (2016). https://doi.org/10.1007/s12010-015-1874-x

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