2.4 Glutarate Fermentation
For shaken flask experiments for glutarate production, single colony was picked into LB and grown overnight at 37°C, 200 rpm. Lately, the cultures at an inoculation volume of 2% were added to the optimized SOB media (previously demonstrated as an effective media for glutarate over-production byE. coli (Zhao, Li, & Deng, 2018a)), grown at 37°C until OD600 ~ 0.6 and then induced by 0.8 mM IPTG(Zhao, Li, & Deng, 2018). The induced cultures were incubated at 30°C, 200 rpm. 4 g/L glucose and 3 g/L or 6 g/L or 9 g/L or 12 g/L acetate were supplemented when necessary, respectively. After inoculation, the flasks were instantly moved to aerobic condition or microaerobic condition controlling by the fermentation bung (Nastasia, 2001) or anaerobic condition using an anaerobic workbench(J.-L. Yu, X.-X. Xia, J.-J. Zhong, & Z.-G. Qian, 2017).
For the fed-batch fermentation, the seed cultures were grown at a rate of 2% into the initial 8 g/L glucose and 6 g/L acetate. The Bgl51464 was cultured at 37°C until the OD600 ~ 0.6. After induction with IPTG, the temperature was reduced to 30°C. And 0.5 vvm, 400 rpm and pH 7.0 were conducted during the whole process. When the glucose was used up, the 50% glycerol was supplemented by pH-stat feeding strategy(Lee, 1996). Samples were taken regularly to detect cell growth and analyze metabolites.