基因组改组技术选育耐酸性琥珀酸放线杆菌

Breeding Actinobacillus succinogenes with Acid-tolerance by Genome Shuffling

摘要:

以琥珀酸产生菌Actinobacillus succinogenes CGMCC 1593为出发菌, 分别经过紫外线-甲基磺酸乙酯(UV-EMS)和紫外线-硫酸二乙酯(UV-DES)诱变处理, 得到7株耐酸性有所提高的突变株。以此作为候选菌库, 经3轮原生质体递进融合, 筛选获得4株可以在pH 5.6下生长的改组菌株。其中改组菌株F3-21在pH 5.6的完全液体培养基中生长的OD值是原始菌的7倍, 在pH 5.2条件下仍能生长; 其摇瓶发酵48 h琥珀酸产量较原始菌株提高48%。在5 L发酵罐中进行分批发酵, 当控制pH在较低值(5.6~6.0)时, F3-21厌氧发酵48 h积累琥珀酸38.1 g/L, 较出发菌株提高了45%; 当控制pH在6.5~7.0时, F3-21厌氧发酵32 h积累琥珀酸40.7 g/L。F3-21在5 L发酵罐中进行补料分批发酵, 厌氧发酵72 h, 产琥珀酸达67.4 g/L。结果说明基因组改组技术能够改进琥珀酸放线菌的耐酸性能及其琥珀酸的产量。

Abstract:

A strain Actinobacillus succinogenes CGMCC 1593 was selected as the parent strain. After UV-EMS and UV-DES treatments respectively, seven mutated 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 both higher yield and acid tolerance were obtained. The shuffled strain namely F3-21 could even survive at pH 5.2. The comparison of the shuffled strains and the parent strain for succinic acid production was also studied here. After 48 h of shake-flask fermentation, the succinic acid concentration of F3-21 was 48% higher than that of the parent strain. When F3-21 was carried out in a 5 liter stirred bioreactor with pH controlled 5.6~6.0, the accumulation of succinic acid in 48 h fermentation attained 38.1 g/L, which was increased by 45% compared with that of the parent strain (26.2 g/L). While pH was controlled at 6.5~7.0, the production of succinic acid in 32 h fermentation attained 40.7 g/L. When F3-21 was carried out in fed-batch fermentation, succinic acid concentration of 67.4 g/L was reached in 72 h fer-mentation. These results indicated that the genome shuffling could improve the acid tolerance and the suc-cinic acid production of A. succinogenes CGMCC 1593.