常压室温等离子体诱变选育L-精氨酸生产菌及发酵条件优化

Breeding and fermentation optimization of L-arginine producing strains

【目的】通过常压室温等离子体诱变技术选育L-精氨酸高产菌株,利用响应面设计探索突变菌株生产L-精氨酸的最佳发酵条件。【方法】采用常压室温等离子体生物诱变系统对实验室保藏的Corynebacterium glutamicum GUI089进行系列诱变,选育L-高精氨酸和8-氮鸟嘌呤抗性菌株。在单因子实验的基础上,应用Plackett-Burman设计从7个因素中筛选出对L-精氨酸合成具有显著效应的(NH4)2SO4、葡萄糖和尿素3个因素。基于上述结果,进一步采用响应面设计优化出主要影响因素的最佳参数水平。【结果】经过一系列的诱变和筛选,选育出一株L-高精氨酸(15 g/L)和8-氮鸟嘌呤(0.7 g/L)抗性菌株,并将此菌株命名为C.glutamicum ARG 3-16。此菌株的L-精氨酸产量比出发菌株提高了49.79%,且发酵液中杂酸的浓度明显降低,特别是L-脯氨酸、L-谷氨酸和L-缬氨酸。在经响应面优化后的最佳发酵条件下,L-精氨酸的产量达到39.72±0.75 g/L,比优化前提高了10.49%。【结论】通过常压室温等离子体诱变技术成功选育出一株L-精氨酸高产菌株,利用响应面法有效地优化了发酵条件,实验结果表明突变株ARG 3-16具有潜在的生产应用价值。

[Objective] To breed L-arginine high-yielding strains by atmospheric and room temperature plasma（ARTP）, and to explore the optimal fermentation conditions of mutants by response surface method. [Methods] The L-homoarginine and 8-azaguanine resistant strain was obtained from the strain Corynebacterium glutamicum GUI089（SGr, L-His-） that was treated by ARTP. Based on the results of single factor experiments, ammonium sulfate, glucose and urea were selected as independent variables to optimize fermentation conditions for L-arginine production from 7 factors by Plackett-Burman design. Subsequently, the levels of the three variables were further optimized by response surface methodology. [Results] After several rounds of screening, a strain was selected resistant to 15 g/L L-homoarginine and 0.7 g/L 8-azaguaine, and designated as C. glutamicum ARG 3-16（L-HAr, 8-AZr, SGr, L-His-）. L-arginine production of ARG 3-16 was 49.79% higher than that of the original strain. Beyond the higher production of L-arginine production, ARG 3-16 accumulated fewer by-products than the original strain, especially the accumulations of L-proline, L-glutamate and L-valine. Under the optimal conditions, the yield of L-arginine reached 39.72±0.75 g/L, 10.49% higher than before optimization. [Conclusion] We successfully bred a high-yielding L-arginine producing strain by ARTP breeding system, and optimized fermentation conditions by response surface methodology, the results suggested that ARG 3-16 was a promising L-arginine producing strain.