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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2021, Vol. 41 Issue (2/3): 38-44    DOI: 10.13523/j.cb.2010005
技术与方法     
等离子体作用结合氧限制模型选育利福霉素SV高产菌株 *
栗波1,2,王泽建3,**(),梁剑光4,**(),刘爱军5,李海东5
1 常熟理工学院生物与食品工程学院 常熟 215500
2 苏州大学医学部药学院 苏州 215500
3 华东理工大学国家生物反应器工程重点实验室 上海 200237
4 常州大学制药与生命科学学院 常州 213164
5 河北欣港药业有限公司 石家庄 051530
Breeding of High-yield Rifamycin SV Strain by Plasma Action Combined with Oxygen Restriction Model
LI Bo1,2,WANG Ze-jian3,**(),LIANG Jian-guang4,**(),LIU Ai-jun5,LI Hai-dong5
1 School of Biological and Food Engineering,Changshu Institute of Technology,Changshu 215500, China
2 College of Pharmaceutical Science,Soochow University,Suzhou 215123, China
3 State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
4 College of Pharmaceutical and Life Sciences, Changzhou University,Changzhou 213164, China
5 Hebei Xingang Pharmaceutical Co., Ltd,Hebei 051530,China
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摘要:

利福霉素SV毒性低、疗效高、抗菌谱广,主要由地中海拟无枝酸菌发酵生产,其发酵过程属于耗氧发酵,供氧直接影响产物形成。为减少发酵过程氧限制影响,进一步提高利福霉素发酵产量,通过构建定向氧限制模型,将常温常压等离子体诱变和无水亚硫酸钠氧限制筛选模型相结合,建立了利福霉素生产菌株24孔板快速培养的高通量筛选方法,高效选育出能够耐受低氧环境的利福霉素SV高产菌株NSMXG-M126,发酵代谢状态参数变化显示,该高产菌株具有更好的氧亲和力。同样的供氧条件下,与对照相比表现出较快的菌体生长速率和利福霉素SV的快速合成能力。在低供氧情况下发酵单位达到7 839mg/L,较出发菌株提高48%,表明耐受低氧的突变菌株具有更高的利福霉素SV生产效率。

关键词: 利福霉素SV地中海拟无枝酸菌高通量筛选ARTP氧限制模型    
Abstract:

Rifamycin SV has low toxicity, high curative effect, and broad antibacterial spectrum. It is mainly produced through the aerobic fermentation by Amycobacterium marinum, and oxygen supply limitation was always the critical factor that affects the rifamycin SV biosynthesis. In order to reduce the influence of oxygen limitation in the fermentation process and further increase the fermentation yield of rifamycin, an oxygen limitation directional model was established with anhydrous sodium sulfite, combining normal temperature and atmospheric pressure plasma mutagenesis(ARTP); a high-throughput screening method of rapid culture of the vinegar-producing strains in 24-well plates was proposed; the high-yielding rifamycin SV strain NSMXG-M126 that can tolerate hypoxia has been efficiently selected. The changes in the parameters of the fermentation metabolic state show that the high-yielding strain has better oxygen affinity. Under the same oxygen supply conditions, it showed a faster bacterial growth rate and rapid synthesis of rifamycin SV compared with the control. In the case of low oxygen supply, the fermentation unit reached 7 839mg/mL, which was 48% higher than the original strain. The results demonstrated that the mutant strains that tolerate hypoxia have higher production efficiency of rifamycin SV.

Key words: Rifamycin SV    Amycobacterium marinum    High-throughput screening    Atmospheric and room temperature plasma (ARTP)    Oxygen-limited model
收稿日期: 2020-11-05 出版日期: 2021-04-08
ZTFLH:  Q815  
基金资助: * 国家重点研发计划资助项目(2017ZX07402003)
通讯作者: 王泽建,梁剑光     E-mail: wzjvictory@163.com;liang4523@126.com
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引用本文:

栗波,王泽建,梁剑光,刘爱军,李海东. 等离子体作用结合氧限制模型选育利福霉素SV高产菌株 *[J]. 中国生物工程杂志, 2021, 41(2/3): 38-44.

LI Bo,WANG Ze-jian,LIANG Jian-guang,LIU Ai-jun,LI Hai-dong. Breeding of High-yield Rifamycin SV Strain by Plasma Action Combined with Oxygen Restriction Model. China Biotechnology, 2021, 41(2/3): 38-44.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2010005        https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I2/3/38

装液量(mL) 1 1.5 2 2.5 3 摇瓶
PMV 28.2±1.3 26.6±1.1 25.2±1.2 23.0±0.9 18.1±1.1 26.4±1.4
单位(mg/L) 4 920±46 5 265±32 5 408±38 4 739±41 2 953±39 5 620±47
比产量(mg/PMV) 174.5±13 197.9±18 214.6±19 206.4±16 163.1±21 212.9±18
表1  初始菌株不同孔板装液量条件下发酵情况及与摇瓶发酵对比
图1  地中海拟无枝酸菌ARTP致死率曲线
无水亚硫酸钠(g/L) 0 0.1 0.2 0.4 0.6 1.0 2.0
CFU 230±15 205±21 190±17 45±4 4±2 0 0
表2  不同无水亚硫酸钠添加环境对菌落生长情况的影响
处理时间(s) 正突变率(%) 负突变率(%)
0 3.8±0.7 4.5±1.0
5 7.5±1.2 13.2±1.4
氧限制筛选模型 15 13.0±1.6 23.8±1.9
25 21.3±2.0 28.5±1.8
35 8.4±1.1 42.0±2.1
正常培养平皿 25 9.1±1.5 22.7±2.2
表3  不同处理时间对氧限制模型条件下正突变率的影响
菌浓
(PMV)
单位
(mg/mL)
比产量
(mg/PMV)
传代1 26.2 6 208.4 237.0
传代2 26.3 6 217.1 236.4
传代3 25.9 6 148.0 237.4
传代4 26.2 6 265.4 239.1
传代5 26.7 6 196.1 232.1
传代6 26.3 6 246.9 237.5
均值 26.3 6 213.7 236.6
表4  菌株遗传稳定性验证
菌株 出发菌株 NSMXG-M126
转速(r/min) 380 380
流量(vvm) 0.6 0.6
OUR[mmol/(L·h)] 10 12
PMV(%) 25 32
效价(mg/mL) 5 659 7 839
比产率(mg/PMV) 188.6 244.9
表5  50L发酵罐中不同供氧条件下出发菌株和NSMXG-M126发酵对比
图2  50L发酵罐中低供氧条件下出发菌株和NSMXG-M126发酵过程对比
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