谷氨酸棒杆菌L-色氨酸营养缺陷型突变 及其高产菌株的选育

Study on breeding of Corynebacterium glutamicum L-tryptophan auxotrophic strains with high yield

中文关键词:  L-色氨酸  营养缺陷型  菌株选育  产酸率  交叉反馈抑制

英文关键词:L-tryptophan, auxotroph  strains selected breeding  acid production rate  cross-feedback inhibition

中文摘要:

      目的 以谷氨酸棒杆菌(Corynebacterium glutamicum)HX-22为出发菌株, 研究了目标发酵产物L-色氨酸合成途径交叉反馈抑制途径代谢突变与色氨酸分解代谢类似物抗性的营养缺陷型突变诱导过程及L-色氨酸高产复合突变菌株的筛选。方法 采用硫酸二乙酯、紫外线、钴60 γ-射线等诱变方法交叉处理起始与突变菌株, 通过营养缺陷表型和自杀代谢底物抗性的筛选方法, 定向突变和选育L-色氨酸合成与分解代谢突变的色氨酸高产菌株。结果 通过多次连续营养缺陷型诱变选育, 筛选出一株具有分支酸-Trp/Phe/Tyr代谢途径L-Phe和L-Tyr合成缺陷型和L-Trp分解代谢自杀代谢底物抗性的L-色氨酸高产菌HX22-118(?Phe--?Tyr--5FTr- 4FPr-SGr); 通过连续传代10次发酵, 色氨酸产酸最高达到28.4 g/L, 平均27.1 g/L, 较出发菌株HX22产酸水平提高81.8%。结论 选育出的高产菌株具有多重代谢途径突变(?Phe--?Tyr--5FTr-4FPr-SGr), 突变体具有良好的遗传稳定性, 具有后续工业化生产应用潜力。

英文摘要:

      Objective To research the breeding of high L-tryptophan yield with multiple synthetic and metabolic pathway mutation strain by using Corynebacterium glutamicum HX-22 as the starting strain. Methods Diethyl sulfate (DES), UV, and cobalt-60 gamma-ray mutagenesis method were performed cross processing starting on C. glutamicum HX-22 and mutant strain. The auxotrophic phenotype and metabolic suicide substrate resistance screening methods were used, and targeted mutagenesis and breeding of L-tryptophan acid synthesis and catabolism mutation of lubricious ammonia acid producing strain. Results Through continuous type defect mutagenesis breeding for many times, the strains with auxotroph and resistance marker of L-tryptophan high yield bacteria HX22-118 (?Phe--?Tyr--5FTr-4FPr-SGr) was screened, then extend the 10 times in succeeding transfer culture, the L-trp fermentation yield reached to 27.1 g/L, which was increased by 81.8% comparing to the wild start strain. Conclusion The selected multiple mutated strain HX22-118 (?Phe--?Tyr--5FTr-4FPr-SGr) with high L-trp yielding strain has a good genetic stability, which can be applied in industrial application.