| L-glutamic acid is one of the chief products of biotechnology. In the process of glutamic acid fermentation, temperature was one of the most important factors directly related to the production efficiency. In order to prevent the loss of bacterial activity in the industrial production of L-glutamic acid, a cooling system has to be employed to remove the heat generated during bacterial fermentation and growth. Therefore, improving the temperature tolerance of the glutamic-acid producing bacterium will reduce the cost for temperature control and thus provide an economic advantage in the industrial production of L-glutamic acid. In the present study, genome shuffling was explored to improve the thermo tolerance and glutamic acid productivity. Through many rounds of genome shuffling, shuffled strains with higher yield and good thermo tolerance were obtained. The fermentation performance of one strain F343 was studied.In this paper, according to the comparison of the temperature characteristic and fermentation performance of the nine strains collected by our laboratory, two strains named SW07-1 and SW07-2 were selected as the initial starting strains. After the characteristic study of the strains, 5 h pre-cultured cells were used in the mutation study. The mutation conditions were as follows: ultraviolet irradiation for 5 min, 1.0 % DES (diethyl sulfate) for 30 min, X-ray treatment for 35 min. Five thermotolerant mutant strains (SW07-1-1, SW07-1-2, SW07-1-3, SW07-4, and SW07-1-5) from SW07-1, which produced more L-glutamic acid than SW07-1 at 38℃were obtained by X-rays, UV, DES mutagenesis, respectively. Three thermotolerant mutant strains (SW07-2-1, SW07-2-2 and SW07-2-3) from SW07-2 were obtained by UV, DES mutagenesis.The optimized conditions for protoplast preparation were as follows: the pretreatment by penicillin sodium is 0.2 u/mL, 2 h (SW07-1) and 1 h (SW07-2) respectively; lysozyme concentration of 1.0 mg/mL; incubation at 37℃for 15 h to allow cell wall lysis. The protoplast inactivation methods were determined to be ultraviolet irradiated for 30 min and heat treatment for 120 min. Under the optimized conditions for protoplast fusion, 35 % PEG 6000, incubation at 32℃for 30 min, the fusion rate was 2.6×10-5.Genome shuffling was carried as A or B procedures. Strains SW07-1-1, SW07-1-2, SW07-1-3, SW07-1-4 and SW-07-1-5 were used as a parent library in recursive protoplast fusion following with flow chart A. After two rounds of genome shuffling, five mutant strains with both high yield at 38℃and good thermotolerance were obtained. The average production of L-glutamic acid of the second shuffled strains was 37 % higher than that of the initial strain SW07-1. In flow chart B, SW07-1-1, SW07-1-2, and SW07-2-1, SW07-2-2, SW07-2-3, which derived from two different initial starting strains SW07-1 and SW07-2 were used as a parent library. Five mutant strains with high yield at 38℃were obtained by three rounds of genome shuffling. The average production of L-glutamic acid of the third shuffled strains reached 56.5 g/L after 40 h of fermentation at 38℃. The production of strain F343 was an increase of 66 % compared to initial strains SW07-1. At 44℃, the strain F343 could grow well while the initial strains couldn't grow.The fermentation performance and thermotolerance of the shuffled strain F343 were significantly improved. Both the production of the L-glutamic acid and the cell density of F343 were higher than these of the initial starting strains from 35℃to 41℃. The best concentration of corn steep liquor for fermentation of F343 strain at 38℃was tested. When using flask feeding glucose fermentation, the production of L-glutamic acid was up to 78 g/L and the conversion rate was up to 55 % after 40 h...
|
PDF Full Text Request