生物技术通报 ›› 2022, Vol. 38 ›› Issue (1): 150-156.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0272

• 研究报告 • 上一篇    下一篇

常温常压等离子体诱变选育高产L-异亮氨酸大肠杆菌

李晓芳(), 刘慧燕, 潘琳, 艾治宇, 李一鸣, 张恒, 方海田()   

  1. 宁夏大学食品与葡萄酒学院 宁夏食品微生物应用技术与安全控制重点实验室,银川 750021
  • 收稿日期:2021-03-09 出版日期:2022-01-26 发布日期:2022-02-22
  • 作者简介:李晓芳,硕士研究生,研究方向:健康食品生物制造;E-mail: 18447051253@163.com
  • 基金资助:
    宁夏回族自治区自然科学基金项目(2020A1170);宁夏食品微生物应用技术与安全控制重点实验室平台建设项目(2018B0008);宁夏食品微生物应用技术与安全控制重点实验室平台建设项目(2019YDDF0062)

Breeding High-yield L-isoleucine Escherichia coli by ARTP Mutagenesis

LI Xiao-fang(), LIU Hui-yan, PAN Lin, AI Zhi-yu, LI Yi-ming, ZHANG Heng, FANG Hai-tian()   

  1. School of Food and Wine,Ningxia Key Laboratory of Food Microbiology Application Technology and Safety Control,Ningxia University,Yinchuan 750021
  • Received:2021-03-09 Published:2022-01-26 Online:2022-02-22

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