常压室温等离子体诱变与微生物微液滴培养选育谷胱甘肽高产菌株

摘要/Abstract

摘要： 为提升毕赤酵母产谷胱甘肽的能力，经常压室温等离子体诱变（Atmospheric and Room Temperature Plasma，ARTP）处理，并结合微生物微液滴培养仪（Microbial Microdroplet Culture System，MMC）驯化，以1,2,4-三氮唑作为筛选标记，获得了一株高产谷胱甘肽的突变株BY-2-24，并对其遗传稳定性进行研究。结果表明：出发菌株经过ARTP诱变处理、抗性梯度平板初筛、MMC适应性进化、摇瓶复筛等，可以选育出高产谷胱甘肽突变株。突变株BY-2-24摇瓶产量可达到312.1 mg/L，是出发菌株的2.34倍，且经过7次传代培养，仍然具有较好的遗传稳定性。研究表明，ARTP与MMC联合使用为谷胱甘肽的高通量选育提供了新的参考，是一种有效的微生物菌种选育方法。

关键词: 谷胱甘肽, 毕赤酵母, 常压室温等离子体诱变, 适应性进化, 菌种筛选

Abstract: In order to improve the ability of Pichia pastoris to produce glutathione, Atmospheric and Room Temperature Plasma (ARTP) treatment was used, combined with Microbial Microdroplet Culture System (MMC) acclimation, to using 1,2,4-triazole as a selection marker, a mutant strain BY-2-24 with high glutathione production was obtained, and its genetic stability was also studied. The results showed that after ARTP mutagenesis treatment, primary screening of resistance gradient plate, MMC adaptive evolution, and re-screening of shake flasks, the starting strains could breed mutants with high glutathione yield, and the yield of BY-2-24 of shake flasks reached 312.1 mg/L, which was 2.34 times than that of the starting strain, and after 7 subcultures, it still has good genetic stability. The research shows that the combined use of ARTP and MMC provides a certain reference for the high-throughput breeding of glutathione, and is an effective method for microbial strain breeding.

Key words: glutathione, Pichia pastoris, atmospheric and room temperature plasma（ARTP）, adaptive evolution, strain selection

凌思雨王洲张会敏李闯刘庆涛刘艳薛正莲. 常压室温等离子体诱变与微生物微液滴培养选育谷胱甘肽高产菌株[J]. 食品科学, 0, (): 0-0.

Huimin Zhang. Breeding of High Yield Glutathione Strain by Atmospheric and Room Temperature Plasma Mutagenesis and Microbial Microdroplet Culture System[J]. FOOD SCIENCE, 0, (): 0-0.

参考文献

[1].参考文献 [2]Do D T H, Fickers P, Ben Tahar I.Improvement of glutathione production by a metabolically engineered Yarrowia lipolytica strain using a small-scale optimization approach[J].Biotechnology Letters, 2021, 43(2):407-414 [3]Tamanna N, Kroeker K, Braun K, et al.The effect of short-term methionine restriction on glutathione synthetic capacity and antioxidant responses at the whole tissue and mitochondrial level in the rat liver[J]. Experimental Gerontology, 2019, 127:110712. DOI:10.1016/j.exger.2019.110712[J].Experimental Gerontology, 2019, 127:1-10 [4]Kresnowati M, Ikhsan N A, Nursa' Adah R S, et al.Evaluation of Glutathione Production Method using Saccharomyces cerevisiae[J].IOP conference series. Materials Science and Engineering, 2019, 543(1):12004-12013 [5]Mezzetti F, De Vero L, Giudici P.Evolved Saccharomyces cerevisiae wine strains with enhanced glutathione production obtained by an evolution-based strategy[J].FEMS yeast research, 2014, 14(6):977-987 [6]蒋秋琪, 吕雪芹, 崔世修, 等.代谢工程改造毕赤酵母发酵生产谷胱甘肽[J].食品与发酵工业, 2020, 46(17):9-14 [7]Zhou W, Yang Y, Tang L, et al.Acrolein-stressed threshold adaptation alters the molecular and metabolic bases of an engineered Saccharomyces cerevisiae to improve glutathione production[J]. Scientific Reports, 2018, 8(1). DOI:10.1038/s41598-018-22836-2[J].Scientific Reports, 2018, 8(1):1-11 [8]王大慧, 聂敏, 卫功元.基于两阶段氨基酸添加的谷胱甘肽发酵高产方法[J].食品科学, 2017, 38(22):22-27 [9]Hamad G M, Taha T H, Alshehri A M, et al.Enhancement of the Glutathione Production by Mutated Yeast[J].Research Journal of Microbiology, 2018, 13(1):28-36 [10]Hamad G M, Taha T H, Alshehri A M, et al..Enhancement of the Glutathione Production by Mutated Yeast Strains and its Potential as Food Supplement and Preservative[J].Research Journal of Microbiology, 2018, 13(1):28-36 [11]Schmacht M, Lorenz E, Senz M.Microbial production of glutathione[J].World Journal of Microbiology and Biotechnology, 2017, 33(6):1-13 [12]Nisamedtinov I, Nisamedtinov I, Kevvai K, et al.Metabolic changes underlying the higher accumulation of glutathione in Saccharomyces cerevisiae mutants[J].Applied microbiology and biotechnology, 2011, 89(4):1029-1037 [13]Schmacht M, Lorenz E, Stahl U, et al.Medium optimization based on yeast's elemental composition for glutathione production in Saccharomyces cerevisiae[J].Journal of Bioscience and Bioengineering, 2017, 123(5):555-561 [14]王红波, 王璐, 孙宪迅, 等.谷胱甘肽发酵生产及其在动物饲料工业中的应用[J].江汉大学学报自然科学版, 2019, 47(04):366-370 [15]Patzschke A, Steiger M G, Holz C, et al.Enhanced glutathione production by evolutionary engineering of Saccharomyces cerevisiae strains[J].Biotechnology journal, 2015, 10(11):1719-1726 [16]梁国斌, 林伟, 汪斌, 等.分批培养产朊假丝酵母合成谷胱甘肽的前体氨基酸优化策略[J].河南工业大学学报自然科学版, 2017, 38(06):62-68 [17]高宇豪, 吴勇杰, 朱亚鑫, 等.产谷胱甘肽毕赤酵母工程菌的构建及能量调控[J].食品与发酵工业, 2021, 47(07):21-26 [18]董永胜, 马蕾, 王艳杰.提高微生物谷胱甘肽产率措施的探讨[J].中国酿造, 2016, 35(05):6-9 [19]郑明坤, 黄家滨, 李友明, 等.诱变结合抗性筛选选育西索米星高产菌株[J].中国抗生素杂志, 2021, 46(01):42-48 [20]金鸣, 王威, 张雪洪.高产洛蒙真菌素洛蒙德链霉菌高通量筛选方法的建立与复合育种[J].微生物学通报, 2020, 47(1):200-209 [21]朱孟峰, 许伟, 邵荣, 等.桑叶中α-葡萄糖苷酶抑制剂产生菌的分离鉴定及诱变选育[J].食品科学, 2017, 38(10):111-116 [22]郭肖杰, 王立言, 张翀, 等.高通量自动化微生物微液滴进化培养与筛选技术及其装备化[J].生物工程学报, 2021, 37(03):991-1003 [23]马钦元, 申雁冰, 丁盼盼, 等.常压室温等离子诱变与微生物微滴培养选育几丁质脱乙酰基酶高产菌株[J].中国酿造, 2020, 39(08):170-174 [24]邓磊, 张豪, 郑穗平.常压室温等离子体诱变与微生物液滴培养系统联用筛选-组氨酸产生菌[J].中国酿造, 2021, 40(02):53-58 [25]曾伟主, 单小玉, 房峻, 等.微液滴适应性进化强化大肠杆菌耐受高浓度-山梨糖[J].食品与发酵工业, 2021, 47(01):1-7 [26]张恩芬, 查飞, 周建宝, 等.酶标仪法测定发酵液中谷胱甘肽的含量[J].滁州学院学报, 2019, 21(05):31-34 [27]钱卫东, 吴启航, 刘昱辰, 等.谷胱甘肽高产酵母融合子的制备及发酵工艺[J].陕西科技大学学报自然科学版, 2017, 35(02):126-130 [28]邹宗胜, 王婧雅, 赵运英, 等.高产纤维素酶突变株的筛选及其产酶条件优化[J].食品科学, 2019, 40(06):48-54 [29]Jian X, Guo X, Wang J, et al.Microbial microdroplet culture system (MMC): An integrated platform for automated,high‐throughput microbial cultivation and adaptive evolution[J].Biotechnology and Bioengineering, 2020, 117(6):1724-1737 [30]Hua X, Wang J, Wu Z, et al.A salt tolerant Enterobacter cloacae mutant for bioaugmentation of petroleum- and salt-contaminated soil[J].Biochemical Engineering Journal, 2010, 49(2):201-206 [31]陈雪, 赵文杰, 冯军, 等.谷胱甘肽产生菌的菌种选育[J].中国医药工业杂志, 2007, 38(7):481-483 [32]冮洁, 单立峰, 吴耘红, 等.值和表面活性剂对酿酒酵母生物合成谷胱甘肽的影响[J].食品科学, 2009, 30(19):223-226