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

• Published in: Applied biochemistry and biotechnology 2016-01, Vol.178 (2), p.324-337
• Main Authors: Zhou, Li, Deng, Can, Cui, Wen-Jing, Liu, Zhong-Mei, Zhou, Zhe-Min
• Format: Article
• Language: English
• Subjects: Aerobiosis; alanine; Alanine - biosynthesis; Alanine Dehydrogenase - genetics; Alanine Dehydrogenase - metabolism; Biochemistry; Bioengineering; Bioreactors; Biotechnology; carbon; Carbon sources; cell growth; Chemistry; Chemistry and Materials Science; Chromosomes; Cultivation; E coli; Escherichia coli; Escherichia coli - genetics; Fermentation; Geobacillus; Geobacillus stearothermophilus; Geobacillus stearothermophilus - enzymology; glucose; Oxygen; production costs; Temperature; thermoplastics; veterinary drugs
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-015-1874-x

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.