常温常压等离子体诱变选育高产L-异亮氨酸大肠杆菌

doi:10.13560/j.cnki.biotech.bull.1985.2021-0272

摘要/Abstract

摘要：

旨在选育L-异亮氨酸高产大肠杆菌。以大肠杆菌K12（Met^-）为出发菌株,经常温常压等离子体（ARTP）诱变,通过微生物高通量液滴培养系统（MMC）筛选,以α-氨基丁酸（α-AB）抗性为筛选标记,得到一株高产L-异亮氨酸的突变菌株大肠杆菌NXU12,并对其遗传稳定性进行了研究。结果表明,出发菌株大肠杆菌K12（Met^-）经过ARTP诱变处理180 s后,通过单因素多水平实验、适应性进化实验、发酵培养复筛、遗传稳定性验证等选育出一株高产L-异亮氨酸诱变大肠杆菌NXU12。该菌株在发酵培养40 h后,L-异亮氨酸产量达到17.462 8 g/L,比原始菌株10.830 9 g/L提高61.23%,且遗传性状稳定。

关键词: 大肠杆菌, ARTP, MMC, α-AB, 抗性标记, L-异亮氨酸

Abstract:

This work aims to breed high-yield L-isoleucine-producing Escherichia coli. Using E. coli K12（Met^-）as the original strain,regular temperature and atmospheric pressure plasma（ARTP）was for mutagenesis,combined with microbial high-throughput droplet culture system（MMC）was for screening,with α-aminobutane acid（α-AB）resistance was used as a selection marker to obtain a mutagenic E. coli NXU12 producing high yield of L-isoleucine and its genetic stability was studied. The results showed that after the original strain K12（Met^-）was subjected to ARTP mutagenesis for 180 s,a strain of high-yield L-isoleucine mutagenesis,E. coli NXU12,was selected through single-factor multi-level experiments,adaptive evolution experiments,fermentation culture re-screening,and genetic stability verification. After 40 h of fermentation and culture,the L-isoleucine yield of this strain reached 17.462 8 g/L,which was 61.23% higher than that by original strain 10.830 9 g/L,and the genetic traits were stable.

Key words: Escherichia coli, ARTP, MMC, α-AB, resistance marker, L-isoleucine

李晓芳, 刘慧燕, 潘琳, 艾治宇, 李一鸣, 张恒, 方海田. 常温常压等离子体诱变选育高产L-异亮氨酸大肠杆菌[J]. 生物技术通报, 2022, 38(1): 150-156.

LI Xiao-fang, LIU Hui-yan, PAN Lin, AI Zhi-yu, LI Yi-ming, ZHANG Heng, FANG Hai-tian. Breeding High-yield L-isoleucine Escherichia coli by ARTP Mutagenesis[J]. Biotechnology Bulletin, 2022, 38(1): 150-156.

图/表 8

图1 大肠杆菌ARTP诱变致死率曲线

Fig. 1 Lethality curve of Escherichia coli ARTP mutagenesis

图2 大肠杆菌NXU12单因素多水平曲线

Fig. 2 Single-factor multi-level curve of E. coli NXU12

图3 大肠杆菌NXU12适应性进化曲线

Fig. 3 Adaptive evolution curve of E. coli NXU12

图4 大肠杆菌K12、NXU12测序结果

Fig. 4 Sequencing results of E. coli K12 and NXU12

图6 大肠杆菌K12、NXU12 L-异亮氨酸浓度

Fig. 6 E. coli K12 and NXU12 L-isoleucine concentration

图5 大肠杆菌K12、NXU12耗糖量曲线

Fig. 5 Sugar consumption curve of E. coli K12 and NXU12

图7 大肠杆菌NXU12传10代后生长曲线

Fig. 7 Growth curve of E. coli NXU12 after 10 generations

图8 大肠杆菌NXU12传10代L-异亮氨酸浓度

Fig. 8 L-isoleucine concentration of E. coli NXU12 for 10 generations

参考文献 21

[1]	王壮壮, 魏佳, 于海波, 等. L-异亮氨酸高产菌选育及其培养基优化[J]. 生物技术通报, 2019, 35(1):82-89. doi: 10.13560/j.cnki.biotech.bull.1985.2018-0634
	Wang ZZ, Wei J, Yu HB, et al. Breeding of strain producing L-isoleucine and medium optimization for it[J]. Biotechnol Bull, 2019, 35(1):82-89.
[2]	Shi F, Fang HM, Niu TF, et al. Overexpression of ppc and lysC to improve the production of 4-hydroxyisoleucine and its precursor l-isoleucine in recombinant Corynebacterium glutamicum ssp. lactofermentum[J]. Enzyme Microb Technol, 2016, 87/88:79-85. doi: 10.1016/j.enzmictec.2016.04.008 URL
[3]	Dong X, Zhao Y, Hu J, et al. Attenuating l-lysine production by deletion of ddh and lysE and their effect on l-threonine and l-isoleucine production in Corynebacterium glutamicum[J]. Enzyme Microb Technol, 2016, 93/94:70-78. doi: 10.1016/j.enzmictec.2016.07.013 URL
[4]	Eggeling L, Morbach S, Sahm H. The fruits of molecular physiology:engineering the l-isoleucine biosynjournal pathway in Corynebacterium glutamicum[J]. J Biotechnol, 1997, 56(3):167-182. doi: 10.1016/S0168-1656(97)00115-6 URL
[5]	Leyval D, Uy D, Delaunay S, et al. Characterisation of the enzyme activities involved in the valine biosynthetic pathway in a valine-producing strain of Corynebacterium glutamicum[J]. J Biotechnol, 2003, 104(1/2/3):241-252. doi: 10.1016/S0168-1656(03)00162-7 URL
[6]	Morbach S, Sahm H, Eggeling L. L-isoleucine production with Corynebacterium glutamicum:further flux increase and limitation of export[J]. Appl Environ Microbiol, 1996, 62(12):4345-4351. doi: 10.1128/aem.62.12.4345-4351.1996 URL
[7]	于丽男. 高产L-异亮氨酸大肠杆菌的选育及其发酵条件优化的研究[D]. 银川:宁夏大学, 2015.
	Yu LN. Breeding of L-isoleucine producing strain and optimization of the fermentation conditions on Escherichia coli[D]. Yinchuan:Ningxia University, 2015.
[8]	Zhang X, Zhang XF, Li HP, et al. Atmospheric and room temperature plasma(ARTP)as a new powerful mutagenesis tool[J]. Appl Microbiol Biotechnol, 2014, 98(12):5387-5396. doi: 10.1007/s00253-014-5755-y pmid: 24769904
[9]	Cheng G, Xu J, Xia X, et al. Breeding L-arginine-producing strains by a novel mutagenesis method:Atmospheric and room temperature plasma(ARTP)[J]. Prep Biochem Biotechnol, 2016, 46(5):509-516. doi: 10.1080/10826068.2015.1084634 URL
[10]	Qiang W, Ling-ran F, Luo W, et al. Mutation breeding of lycopene-producing strain Blakeslea trispora by a novel atmospheric and room temperature plasma(ARTP)[J]. Appl Biochem Biotechnol, 2014, 174(1):452-460. doi: 10.1007/s12010-014-0998-8 pmid: 24903962
[11]	Li X, Liu R, Li J, et al. Enhanced arachidonic acid production from Mortierella alpina combining atmospheric and room temperature plasma(ARTP)and diethyl sulfate treatments[J]. Bioresour Technol, 2015, 177:134-140. doi: 10.1016/j.biortech.2014.11.051 URL
[12]	Zhang C, Qin J, Dai Y, et al. Atmospheric and room temperature plasma(ARTP)mutagenesis enables xylitol over-production with yeast Candida tropicalis[J]. J Biotechnol, 2019, 296:7-13. doi: S0168-1656(19)30068-9 pmid: 30853634
[13]	Bajorath J. Integration of virtual and high-throughput screening[J]. Nat Rev Drug Discov, 2002, 1(11):882-894. pmid: 12415248
[14]	Morbach S, Burger U, Peter H, et al. Activity regulation of osmoprotectant transporters in Corynebacterium glutamicum[J]. Comp Biochem Physiol A:Mol Integr Physiol, 2000, 126:83.
[15]	Wang XY. Strategy for improving L-isoleucine production efficiency in Corynebacterium glutamicum[J]. Appl Microbiol Biotechnol, 2019, 103(5):2101-2111. doi: 10.1007/s00253-019-09632-2 URL
[16]	李燕军, 张海宾, 麻杰, 等. 代谢工程改造大肠杆菌合成L-异亮氨酸的研究[J]. 发酵科技通讯, 2016, 45(3):133-139.
	Li YJ, Zhang HB, Ma J, et al. Study on metabolic engineering of Escherichia coli for L-isoleucine production[J]. Bull Ferment Sci Technol, 2016, 45(3):133-139.
[17]	沈加彬, 胡荣涛, 罗磊, 等. 响应面法优化L-异亮氨酸产生菌的摇瓶发酵条件[J]. 福建师范大学学报:自然科学版, 2018, 34(2):48-56.
	Shen JB, Hu RT, Luo L, et al. Optimization the Flask-shaking fermentation conditions of an L-isoleucine producing strain Brevibacterium flavum by response surface method[J]. J Fujian Norm Univ:Nat Sci Ed, 2018, 34(2):48-56.
[18]	陈胜杰, 高翔, 袁戎宇. 常温常压等离子诱变结合玉米秸秆水解液驯化酿酒酵母生产生物乙醇[J]. 食品与发酵工业, 2020, 46(4):167-171.
	Chen SJ, Gao X, Yuan RY. Screening of high-yield ethanol Saccharomyces cerevisiae by the combination of atmospheric and room temperature plasma with corn stalk hydrolysate[J]. Food Ferment Ind, 2020, 46(4):167-171.
[19]	范新蕾, 肖成建, 顾秋亚, 等. ARTP诱变选育葡萄糖氧化酶高产菌株及发酵条件优化[J]. 工业微生物, 2015, 45(1):15-19.
	Fan XL, Xiao CJ, Gu QY, et al. ARTP mutation breeding of glucose oxidase-producing strains and optimization of fermentation conditions[J]. Ind Microbiol, 2015, 45(1):15-19.
[20]	孔帅, 陈敏, 郑美娟, 等. 常温常压等离子体诱变选育高产L-异亮氨酸谷氨酸棒杆菌[J]. 中国酿造, 2019, 38(7):76-79.
	Kong S, Chen M, Zheng MJ, et al. Mutation breeding of high-yield L-isoleucine Corynebacterium glutamicum by atmospheric and room temperature plasmas mutagenesis[J]. China Brew, 2019, 38(7):76-79.
[21]	Sun X, Li QG, Wang Y, et al. Isoleucyl-tRNA synthetase mutant based whole-cell biosensor for high-throughput selection of isoleucine overproducers[J]. Biosens Bioelectron, 2021, 172:112783. doi: 10.1016/j.bios.2020.112783 URL