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乙基伊维菌素高产菌株选育及其发酵工艺优化

李冰 齐欢 张成宏 张绍勇 张立钦 向文胜 王继栋

李冰, 齐欢, 张成宏, 张绍勇, 张立钦, 向文胜, 王继栋. 乙基伊维菌素高产菌株选育及其发酵工艺优化[J]. 农药学学报. doi: 10.16801/j.issn.1008-7303.2022.0070
引用本文: 李冰, 齐欢, 张成宏, 张绍勇, 张立钦, 向文胜, 王继栋. 乙基伊维菌素高产菌株选育及其发酵工艺优化[J]. 农药学学报. doi: 10.16801/j.issn.1008-7303.2022.0070
LI Bing, QI Huan, ZHANG Chenghong, ZHANG Shaoyong, ZHANG Liqin, XIANG Wensheng, WANG Jidong. Breeding of high-yield strains for producing ethyl ivermectin and the optimization of the fermentation process[J]. Chinese Journal of Pesticide Science. doi: 10.16801/j.issn.1008-7303.2022.0070
Citation: LI Bing, QI Huan, ZHANG Chenghong, ZHANG Shaoyong, ZHANG Liqin, XIANG Wensheng, WANG Jidong. Breeding of high-yield strains for producing ethyl ivermectin and the optimization of the fermentation process[J]. Chinese Journal of Pesticide Science. doi: 10.16801/j.issn.1008-7303.2022.0070

乙基伊维菌素高产菌株选育及其发酵工艺优化

doi: 10.16801/j.issn.1008-7303.2022.0070
基金项目: 浙江省重点研发计划 (2020C02028).
详细信息
    作者简介:

    李冰,1026794936@qq.com

    通讯作者:

    王继栋,02752@zjhu.edu.cn

  • 中图分类号: TQ458.1

Breeding of high-yield strains for producing ethyl ivermectin and the optimization of the fermentation process

Funds: the Key Research and Development Project Programs of Zhejiang Province (2020C02028)
  • 摘要: 基因工程菌Streptomyces avermitilis AVE-H39产生的甲基和乙基伊维菌素 (methyl and ethyl ivermectins),为新型高效低毒的阿维菌素 (avermectin) 衍生物,具有很好的开发应用前景。活性测试结果显示,乙基伊维菌素对朱砂叶螨Tetranychus cinnabarinus和松材线虫Bursaphelenchus xylophilus等农林作物害虫的活性优于甲基伊维菌素。鉴于菌株S. avermitilis AVE-H39的乙基伊维菌素产量低,制约了产业化开发,本研究经过多轮诱变选育,筛选获得了一株乙基伊维菌素发酵单位高的突变菌株,并通过Plackett-Burman 试验设计及Box-Behnken 设计-响应曲面法 (response surface methodology, RSM)对该突变菌株的发酵培养基进行改良。经优化后的发酵条件为:玉米淀粉149.8 g/L,黄豆粉38.1 g/L,(NH4)2SO4 3.04 g/L,甘露醇30.0 g/L,酵母抽提物20.0 g/L,CaCO3 3.0 g/L,CoCl2 0.01 mg/L,FeSO4 0.002 mg/L,转速220 r/min,pH 7.2,装液量30 mL/250 mL,接种量5%,发酵温度28 ℃。在此条件下,乙基伊维菌素的发酵效价达到最大值,为4 965 mg/L。研究结果将为开发以乙基伊维菌素为主成分的新型十六元大环内酯农药奠定基础。
  • 1  甲基伊维菌素和乙基伊维菌素结构式

    1.  The structures of methyl and ethyl ivermectin

    图  1  UV诱变时间对S. avermitilis AVE-H39菌株孢子致死率和正突变率的影响

    Figure  1.  Effect of UV irradiation time on spore lethal rate and positive mutation rate of S. avermitilis AVE-H39 strain

    图  2  ARTP诱变时间对S. avermitilis AVE-H39 菌株孢子致死率和正突变率的影响

    Figure  2.  Effect of ARTP irradiation time on spore lethal rate and positive mutation rate of S. avermitilis AVE-H39 strain

    图  3  NTG质量浓度对S. avermitilis AVE-H39菌株孢子致死率和正突变率的影响

    Figure  3.  Effect of NTG concentration on spore lethal rate and positive mutation rate of S. avermitilis AVE-H39 strain

    图  4  EMS浓度对S. avermitilis AVE-H39菌株致死率和正突变率的影响

    Figure  4.  Effect of EMS concentration on spore lethal rate and positive mutation rate of S. avermitilis AVE-H39 strain

    图  5  选育前后菌落形态对比

    A:原始菌株;B:高产菌株。

    Figure  5.  Colony morphology before and after strain improvement

    A: Original strain; B: High yield strain.

    图  6  选育前后摇瓶发酵HPLC检测结果对比

    A:原始菌株;B:高产菌株。

    Figure  6.  HPLC chromatograms of the shake-flask fermentation broths before and after strain improvement

    A: Original strain; B: High yield strain.

    图  7  速效碳源种类对发酵效价的影响

    Figure  7.  Effect of available carbon sources on fermentation titer

    图  8  迟效碳源种类对发酵效价的影响

    Figure  8.  Effect of delayed carbon sources on fermentation titer

    图  9  速效氮源种类对发酵效价的影响

    Figure  9.  Effect of available nitrogen sources on fermentation titer

    图  10  迟效氮源种类对发酵效价的影响

    Figure  10.  Effect of delayed nitrogen sources on fermentation titer

    图  11  无机盐种类对发酵效价的影响

    Figure  11.  Effect of inorganic salts on fermentation titer

    图  12  微量元素种类对发酵效价的影响

    Figure  12.  Effect of trace elements on fermentation titer

    图  13  玉米淀粉与黄豆粉对乙基伊维菌素效价的交互影响

    I:等高线图;II:响应面图。

    Figure  13.  Interaction effect of corn starch and soybean powder on the titer of ethyl ivermectin

    I: Contour map; II: Response surface diagram.

    图  14  玉米淀粉与(NH4)2SO4对乙基伊维菌素效价的交互影响

    I:等高线图;II:响应面图。

    Figure  14.  Interaction effect of corn starch and (NH4)2SO4 on the titer of ethyl ivermectin

    I: Contour map; II: Response surface diagram.

    图  15  黄豆粉与(NH4)2SO4对乙基伊维菌素效价的交互影响

    I:等高线图;II:响应面图。

    Figure  15.  Interaction effect of soybean powder and (NH4)2SO4 on the titer of ethyl ivermectin

    I: Contour map; II: Response surface diagram.

    图  16  发酵代谢相关参数变化曲线

    Figure  16.  Variation curves of metabolism-relevant parameters during the fermentation process

    表  1  Plackett-Burman试验设计因素与水平

    Table  1.   Factors and levels in Plackett-Burman experiment

    编号
    Symbol
    因素
    Factor
    水平 Level
    −101
    A 玉米淀粉 Corn starch/(g/L) 90 120 150
    B 甘露醇 Mannitol/(g/L) 15 20 25
    C 酵母抽提物 Yeast extract/(g/L) 15 20 25
    D 虚拟 Dummy
    E 黄豆粉 Soybean powder/(g/L) 20 30 40
    F CaCO3 /(g/L) 2 3 4
    G (NH4)2SO4/(g/L) 1 2 3
    H 虚拟 Dummy
    J FeSO4 /(g/L) 0.001 0.002 0.003
    K CoCl2 /(g/L) 0.001 0.002 0.003
    L pH 6.5 7.0 7.5
    下载: 导出CSV

    表  2  Plackett-Burman试验结果

    Table  2.   Results of Plackett-Burman experiment

    序号
    Run number
    因素 Factor乙基伊维菌素
    ethyl ivermectin/
    (mg/L)
    ABCEFGJKL
    1−1−1−1−1−1−1−1−1−13370
    2−111111−1−114069
    311−1−11−11−113906
    411−111−1−11−14212
    5−11−11−111−1−13825
    61−1−11−11−1114618
    71−11−111−1−1−14251
    81−111−1−11−114194
    9−1−1−1−1111113864
    10−1−1111−111−13847
    11−111−1−1−1−1113590
    12111−1−1111−14232
    下载: 导出CSV

    表  3  Plackett-Burman试验结果的方差分析

    Table  3.   ANOVA analysis of Plackett-Burman design

    编号
    Symbol
    因素
    Factor
    均平方
    Mean square
    F
    F value
    P
    P value
    显著性
    Prominence
    A 玉米淀粉 Corn starch/(g/L) 774192 102.51 0.0096 **
    B 甘露醇 Mannitol/(g/L) 1008.3 0.13 0.7498 NS
    C 酵母抽提物 Yeast extract/(g/L) 28812 3.82 0.1900 NS
    E 黄豆粉 Soybean powder/(g/L) 255792 33.87 0.0283 *
    F CaCO3 1200 0.16 0.7287 NS
    G (NH4)2SO4 197633.3 26.17 0.0362 *
    J FeSO4 16280.3 2.16 0.2798 NS
    K CoCl2 25025.3 3.31 0.2103 NS
    L pH 7701.3 1.02 0.4189 NS
    注:“*”代表显著(P<0.05),“**”代表极显著(P<0.01),“NS”代表不显著。Note: "*" means significance (P<0.05), "**" means extreme significance (P<0.01), and "NS" means no significance.
    下载: 导出CSV

    表  4  最陡爬坡试验结果

    Table  4.   The corresponding responses of the steepest ascending experiment

    序号
    Run
    number
    A: 玉米淀粉
    Corn
    starch/
    (g/L)
    E: 黄豆粉
    Soybean
    powder
    (g/L)
    G: (NH4)2SO4/
    (g/L)
    乙基伊维菌素产量
    Yield of ethyl
    ivermectin/
    (mg/L)
    11202024512
    21403034836
    31604044781
    41805054129
    下载: 导出CSV

    表  5  Box-Behnken试验因素水平及编码

    Table  5.   Factors and levels in Box-Behnken experiment design

    编号
    Symbol
    因素
    Factor
    水平 Level
    −101
    A 玉米淀粉 Corn starch /(g/L) 120 140 160
    B 黄豆粉 Soybean powder/(g/L) 20 30 40
    C (NH4)2SO4/(g/L) 2 3 4
    下载: 导出CSV

    表  6  Box-Benhnken试验设计与结果

    Table  6.   Design and results of Box-Benhnken experiment

    序号
    Run
    number
    A: 玉米淀粉
    Corn
    starch/
    (g/L)
    B: 黄豆粉
    Soybean
    powder/
    (g/L)
    C: (NH4)2SO4/
    (g/L)
    乙基伊维菌素产量
    Yield of ethyl
    ivermectin/
    (mg/L)
    1−1−104289
    20004870
    30−1−14506
    40−114595
    50004803
    610−14706
    71104962
    8−1014451
    9−1104580
    101014645
    110004895
    12−10−14416
    1301−14676
    140114776
    151−104631
    下载: 导出CSV

    表  7  回归模型的方差分析

    Table  7.   ANOVA analysis for response surface quadratic model

    来源
    Source
    平方和
    Sum of squares
    自由度
    Degrees of freedom
    均方
    Mean square
    F
    F value
    P
    P value
    显著性
    Prominence
    模型 Model475 700952 895.269.90.010 6*
    A182 4001182 40034.180.002 1**
    B118 3001118 30022.170.005 3**
    C3 321.113 321.10.620.465 9NS
    AB400.01400.00.070.795 2NS
    AC2 304.012 304.00.430.540 2NS
    BC30.3130.30.010.942 9NS
    A297 050.5197 050.518.180.008 0**
    B222 680.5122 680.54.250.094 3NS
    C271724.5171 724.513.440.014 5NS
    残差 Residual26 684.2555 336.85
    失拟 Lack of fit22 158.2537 386.0833.260.243 3NS
    纯误差 Pure error452622263
    总离差 Cor total502 40014
    注:“*”代表显著(P<0.05),“**”代表极显著(P<0.01),“NS”代表不显著。Note: "*" means significance (P<0.05), "**" means extreme significance (P<0.01), and "NS" means no significance.
    下载: 导出CSV
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出版历程
  • 收稿日期:  2022-04-06
  • 录用日期:  2022-06-23
  • 网络出版日期:  2022-08-04

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