常压室温等离子体诱变高通量筛选N-乙酰氨基葡萄糖高产突变株

High-throughput screening of Bacillus subtilis for N-acetylglucosamine production by ARTP mutation technology

采用新型常压室温等离子体(ARTP)诱变产N-乙酰氨基葡萄糖工程菌BSGN6,并应用显色法和96孔板培养方法筛选N-乙酰氨基葡萄糖高产突变株。研究表明显色反应时添加四硼酸钾溶液1μL、PDABA 125μL、反应时间5 min、反应温度96℃时,N-乙酰氨基葡萄糖检测的准确度最高。通过多轮ARTP诱变及高通量筛选最终获得突变株(4A12),3 L罐发酵Glc NAc产量达到36.14 g/L,较出发菌株(BSGN6)提高了65.32%。经过50次传代后性状稳定。ARTP诱变技术作为获得产N-乙酰氨基葡萄糖高产菌株的有效途径,与分子生物学手段相比,本方法更加快捷高效。

A novel atmospheric and room temperature plasma （ARTP） mutagenesis system was used by combining with the high throughput screening of 96-well mieroplates and chromogenic reaction to obtain high-yielding mutants. The optimum conditions for chromogenic reaction were as follows： otassium tetraborate solution 1μL, PDABA 125 μL, reaction time 5 min, temperature 96℃. Through several rounds of ARTP mutation and high-throughput screening, a mutant （4A12） was obtained. In 3 L fermentor, the maximum GlcNAc production reached 36.14 g/L, which improved 65.32% compared with that of the control strain BSGN6. The results of subculture test showed that the mutant strain had stable hereditary characteristics after 50 generations. ARTP mutagenesis technique as an effective way to obtain highyielding GlcNAc strains was better than molecular biology methods.