| Succinic acid, as an important platform of C4 compounds, is widely used inpharmaceutical, food, biodegradable plastics and chemical industry. Fermentative productionof succinic acid is a kind of low carbon, clean and green process in line with the requirementsof sustainable development. In this work, breeding of high succinic acid-producing strainusing Genome shuffling was studied. The mutant libraries derived from Actinobacillussuccinogenes F3-21 were obtained by mutagenesis of acridine yellow, ultraviolet, UV-EMSand NTG treatments respectively. High-yielding mutants were screened with the procedure of"cultured in 96-well plates, concentrated assay by HPLC and fermentation in anaerobicbottle". Through three rounds of Genome shuffling, a shuffed strain F3-Ⅱ-3-F (namely F3-ZK)was obtained with the highest yield production increasing by 41.8%. And the fermentativecharacteristics of shuffled strains were also investigated.Firstly, a screen procedure of"cultured in 96-well plates, concentrated detection byHPLC and fermentation in anaerobic bottle"was established. By A. succinogenes F3-21mutagenized with acridine yellow, ultraviolet, UV-EMS and NTG respectively, there were got1056 colonies. In which 11 high-yielding mutants III-9-H, IV-7-A, IV-7-C,Ⅴ-12-B,Ⅵ-10-C,VII-11-H, VIII-10-H, IX-2-C,Ⅹ-8-E,Ⅺ-8-B, andⅫ-7-B were obtained by this screenprocedure. The average succinic acid production of these 11 mutants was increased by 13.9%compared to that of F3-21 in shake flask. Four strains named F1-I-3-F, F1-II-7-B, F1-III-2-Eand F1-IV-9-D were obtained by first round of Genome shuffling of these 11 mutants, whichthe average succinic acid production in shake flask was increased by 20.2% compared toF3-21. Four F2 shuffled strains F2-1-5-B, F2-II-5-H, F2-III-6-D and F2- III-10-F were screenedafter the second round of Genome shuffling using the F1 as parents and the average ofsuccinic acid production in shake flask was increased by 32.9% compared to F3-21. After thethird round of Genome shuffling screening three F3 strains F3-Ⅰ-9-C, F3-Ⅱ-3-F andF3-Ⅱ-10-D were selected, the average the average of succinic acid production in shake flaskwas increased by 45.7% compared to F3-21. In which F3-Ⅱ-3-F could produce succinic acidconcentration reached 38.9 g/L that was increased by 47.6% compared to F3-21 in shake flask.When inoculations were passaged in succession 10 generations, the succinic acid productionkeeps stabilization.The results of succinic acid production scaled up in 5 L stirred fermenter were asfollowing: for F1-Ⅳ-9-D succinic acid concentration attained to 88.5 g/L after 72 h fed-batchfermentation, and the productivity and yield were 1.23 g/(L·h) and 0.67% correspondingly.F2-Ⅲ-6-D succinic acid concentration attained to 87.3 g/L after 48 h in fed-batch fermentation,and the productivity and yield were 1.82 g/(L·h) and 0.71% correspondingly. F3-Ⅱ-3-Fsuccinic acid concentration attained to 95.6 g/L after 48 h in fed-batch fermentation, and theproductivity and yield were 1.99 g/(L·h) and 0.71% correspondingly, the succinic acidproduction was increased by 47.6%, the productivity increased by 111.7% and the yieldincreased by 9.2% compared to those of F3-21, respectively. The shuffled strain F3-Ⅱ-3-F and original strain F3-21 glucose metabolic flux andmetabolic pathways enzyme activity were analyzed. Compared with F3-21, the metabolic fluxto succinic acid of F3-Ⅱ-3-F was increased by 31.6%, while the main byproduct acetic acidwere decreased by 67.8%. The specific activities of hexokinase and phosphoenolpyruvatecarboxylase kinase were increased by 15.4% and 48.5% respectively, while the pyruvatekinase activity, acetate kinase specific activity and phosphate transfer to acetyl enzymespecific activity were decreased by 21.7%, 14.9% and 6.2% respectively. The results reflectedthat the higher yield of F3-II-3-F may be due to the change of these enzymatic activities. |
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