| Succinic acid, as a carbon-intermediate chemical, can be used as a precursor of numerous products, including pharmaceuticals, food additives, green solvents, and biodegradable polymers. Being based on renewable resources, succinic acid produced by microbial fermentation has good environmental benefits and attracted great interest currently. In the present study, genome shuffling was explored to improve the acid-tolerance and succinic acid productivity of Actinobacillus succinogenes. A strain Actinobacillus succinogenes CGMCC 1593 was selected as the parent strain. After UV-EMS and UV-DES treatments respectively, seven mutant strains with subtle improvements in acid tolerance were obtained, and were subjected for recursive protoplast fusion. Through three rounds of genome shuffling, four shuffled strains with higher yield and good acid tolerance were obtained. The fermentation performance of one strain F3-21 was studied.After the characteristic study of strain CGMCC 1593, the critical pH of screening plate and liquid screening medium is pH 5.6. 12 h pre-cultured cells were used in the mutation study and protoplast preparation. The mutation conditions were as follows, UV-EMS treatment: UV for 20 s and 2.0% EMS for 30 min; UV-DES treatment: UV for 20 s and 0.8% EMS for 20 min. The mutant strains with high yield, namely UE-7,UE-10,UE-12,UE-16,UD-6,UD-11,UD-15, were obtained by UV-EMS and UV-DES mutagenesis, respectively.The optimized conditions for protoplast preparation,regeneration and fusion were as follows: adding 0.4% (v/v) glycine into medium before enzymolysis; lysozyme concentration of 0.1 mg/mL; incubation at 37℃for 30 min to allow cell wall lysis; hypertonic phosphate buffer and double-lay plate for regeneration.Under the optimized conditions, incubation at 37℃for 5 min for protoplast fusion, the fusion rate was 2.78×10-5.Four mutant strains namely F3-5,F3-9,F3-14 and F3-21 with high yield and good acid tolerance were obtained by three rounds of genome shuffling. After 48 h of shake-flask fermentation, the succinic acid concentration of the shuffled strain named F3-21 ( 34 g/L) was 48% higher than that of the parent strain. The comparison of the shuffled and parent strains for acid tolerance was also studied. The shuffled strain F3-21 could even survive at pH 5.2. At pH 5.8 and pH 5.6, the cell density of F3-21 was 2.5 and 7 times higher than the parent strain respectively.The primary study on fermentation performance of the strain F3-21 was carried out in a 5 liter stirred bioreactor. When pH was controlled at 5.6-6.0 by MgCO3 , the accumulation of succinic acid in 48 h fermentation attained 38.1 g/L, which was an increase of 45% compared with that of the parent strain ( 26.2 g/L ). The total consumption of MgCO3 also decreased by 22.5% compared with that of the parent strain. When F3-21 was carried out in fed-batch fermentation,, succinic acid concentration of 67.4g/L was reached in 72h fermentation.When pH was controlled at 6.5 by Na2CO3, the production of succinic acid in 32 h fermentation attained 41.5 g/L, succinic acid yield was 83%, excelled controlling pH at 6.5-7.0 by MgCO3 (succinic acid 40.7 g/L, succinic acid yield 81.4%). It indicated that Na2CO3 could be used as a substitute for MgCO3 to pH-control during F3-21 fermentation, thus reducing the production cost.
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