3.1 Glutarate tolerance and degradation test
It has generally been believed that glutarate was toxic to any microorganism, and different strains had different tolerances to toxicity(J. Adkins, J. Jordan, & D. R. Nielsen, 2013; W. Li et al., 2019; Rohles et al., 2018). Although many studies have been done on its physiological roles(J. Adkins, J. Jordan, & D. R. Nielsen, 2013; W. Li et al., 2019; Rohles et al., 2018), glutarate tolerance remained unclear. Thus, glutarate tolerance was first performed before production. The tolerance examination was conducted in an optimized SOB medium(Zhao, Li, & Deng, 2018) with Bgl4146 at 37 °C, which was also used in glutarate production. The cells in exponential growth were placed into increasing glutarate concentrations (Fig. 2A). After cultivating to 30 g/L glutarate, cells grew well although the growth curve was lower than the control (no glutarate). When the concentration of glutaric acid reached 50 g/L, the cells still grew slowly. However, the cells hardly grew and even some cell lysis was discovered when the glutarate concentration increasing to 100 or 150 g/L. Therefore, at least 50 g/L glutarate was able to be tolerated by Bgl4146, this was verified in fermentation production of glutaric acid(W. Li et al., 2019).
Then we also tested the glutarate degradation in Bgl4146 because of theCsiD and YgaF in E. coli involving in the utilization of glutarate(Zhang, Gao, Guo, Guo, Kang, Xiao, Yan, Tao, Zhang, & Dong, 2018). The Bgl4146 was detected in M9 with 5 g/L glutarate as the single carbon source for glutarate degradation (Fig. 2B). However, the strain hardly grew and consumed only 0.1% glutarate in 96 h (Fig. 2B). Therefore, we would not consider the degradation of glutaric acid in the next study.