Actinobacillus Succinogenes Breeding Of Actinobacillus Succinogenes By Microbial Genome Shuffling

In this thesis, Na+-tolerance characteristic of Actinobacillus succinogenes CGMCC 1593 isolated by our laboratory was studied. Three Na+-tolerance mutant strains (X-8, UV-17, SE-6), were obtained by X-ray, ultraviolet irradiation and EMS mutagenesis respectively. These mutant strains, as well as strain SF-9 which was obtained by NTG mutagenesis in previous work were used as a parent library in recursive fusion. Through there rounds of genome shuffling, four mutant strains with high yield and good Na+-tolerance were obtained, and the fermentation performance of one strain F3-10 was studied.After the characteristic study of strain SW0580, screening plate containing 0.8 mol/L NaCl and 5 h pre-cultured cells were used in the mutation study. The mutation conditions were as follows: X-ray treatment for 40 min, ultraviolet irradiation for 20 s, 2.0% EMS for 20 min. The mutant strains with high yield, namely X-8, UV-17 and SE-6, were obtained by X-ray, ultraviolet irradiation and EMS mutagenesis, respectively.The optimized conditions for protoplast preparation and regeneration were as follows: the cells being cultured for 5 h; lysozyme concentration of 0.1 mg/mL; incubation at 37℃for 30 min to allow cell wall lysis; 0.3 mol/L of sucrose as osmotic stabilizer; and double-lay plate for regeneration. The protoplast inactivation methods were determined to be ultraviolet irradiation for 5 min and heat treatments for 25 min. Under the optimized conditions for protoplast fusion, 40% PEG 6000, incubation at 37℃for 2 min, the fusion rate was 2.78×10-5.Four mutant strains namely F3-2, F3-10, F3-13 and F3-15 with high yield were obtained by three rounds of genome shuffling. Using strain F3-10, succinic acid concentration of 39.7 g/L and 30.3 g/L were obtained after 48 h of fermentation in medium containing 0 and 0.2 mol/L NaCl in anaerobic bottles respectively, this is an increase of 36.0% and 72.1% respectively. When batch fermentation was conducted using Na2CO3 for pH control in 5-L bioreactor using cane molasses, the production of succinic acid in 48 h(40.6 g/L)was increased by 56.2% compared with that of wild strain (26.8 g/L). The fermentation performance and Na+-tolerance of the mutant strain F3-10 were significantly improved, and F3-10 showed genetic stability.The primary study on fermentation performance of the strain F3-10 was carried out. Batch fermentation in 5-L bioreactor using cane molasses, maltose syrup and glucose as carbon sources was conducted, highest succinic acid yield was observed using cane molasses as carbon source. The effect of yeast extract and corn steep liquor on succinic acid fermentation was also investigated. The results showed that succinic acid yield decreased evidently without yeast extract, and the yield of succinic acid increased with the addition of corn steep liquor. Fermentation using maltose syrup in a 25 L bioreactor was also carried out, and the final succinic acid concentration of 33.2 g/L was achieved at 48 h, which was comparable with that in 5-L bioreactor.