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  • CN 31-1691/TQ

利用CRISPR/Cas9系统构建低DCPC杂质含量的CPC工业高产菌种

徐燕 冯涛 储炬

徐燕, 冯涛, 储炬. 利用CRISPR/Cas9系统构建低DCPC杂质含量的CPC工业高产菌种[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20200623003
引用本文: 徐燕, 冯涛, 储炬. 利用CRISPR/Cas9系统构建低DCPC杂质含量的CPC工业高产菌种[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20200623003
XU Yan, Tao Feng, CHU Ju. Construction of CPC high-yield industrial strain with low DCPC content using CRISPR / Cas9 system[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.20200623003
Citation: XU Yan, Tao Feng, CHU Ju. Construction of CPC high-yield industrial strain with low DCPC content using CRISPR / Cas9 system[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.20200623003

利用CRISPR/Cas9系统构建低DCPC杂质含量的CPC工业高产菌种

doi: 10.14135/j.cnki.1006-3080.20200623003
详细信息
    作者简介:

    徐燕:徐 燕(1995—),女,浙江绍兴人,硕士生,主要研究方向:工业微生物基因工程改造。E-mail:yanxu_ecust@163.com

    通讯作者:

    储 炬,E-mail:juchu@ecust.edu.cn

  • 中图分类号: Q815

Construction of CPC high-yield industrial strain with low DCPC content using CRISPR / Cas9 system

  • 摘要: 以头孢菌素C(CPC)工业生产菌株顶头孢霉1-D1为研究对象,利用CRISPR/Cas9基因编辑技术降低杂质脱乙酰头孢菌素C(DCPC)累积,提高CPC产量与质量。首先,构建了CPC乙酰水解酶敲除菌株Ac-ΔcahB以期减少CPC水解。然后通过导入含cefG基因表达盒的Donor DNA,构建Ac-ΔcahB::cefG菌株以期提高DCPC转化效率。通过摇瓶发酵验证表明Ac-ΔcahB效价未有提高仅杂质含量略有下降,而Ac-ΔcahB::cefG不仅产量比对照提高约32.6%,达6 072 μg/mL,且杂质含量显著降低,为6.81%。从cefG转录水平来看,Ac-ΔcahB::cefG菌株在96 h转录量提高了5倍。可以发现,cefG基因在强启动子PgpdA作用下可提高表达量,促进DCPC转化,从而提高CPC产量。此外,在工业顶头孢霉中,结合Donor DNA共同作用的CRISPR/Cas9系统是一个更为高效的基因编辑方法。

     

  • 图  1  载体pAN7-cahB构建电泳图。

    Figure  1.  The electrophoretogram of pAN7-cahB construction process. Fig A: cahB-sgRNA fragment (1:84 bp;2:204 bp;3:288 bp); Fig B: pAN7-sorA digested by HindIII and SpeI

    图  2  pAN7-1-cefG构建电泳图。

    Figure  2.  Electrophoresis map of the pAN7-1-cefG construction.

    图  3  Donor DNA构建流程图

    Figure  3.  Donor DNA generation flow chart

    图  4  Donor DNA构建电泳图。

    Figure  4.  Donor DNA construction electrophoretograms.

    图  5  原生质体转化实验流程图

    Figure  5.  The flow chart of protoplast transformation experiment

    图  6  Ac-ΔcahB转化子验证结果。

    Figure  6.  Ac-ΔcahB transformant validation results.

    图  7  Ac-ΔcahB::cefG验证原理图

    Figure  7.  Ac-ΔcahB::cefG verification principle diagram

    图  8  Ac-ΔcahB::cefG PCR验证图。

    Figure  8.  Ac-ΔcahB::cefG PCR verification chart.

    图  9  出发菌株与突变株CPC产量比较。数据代表三个重复试验的平均值和标准偏差。使用单因素方差分析对顶头孢霉1-D1与两个突变株的CPC产量差异进行统计学意义比较(***p <0.001; **p < 0.01; *p < 0.05)。

    Figure  9.  CPC production comparison of starting strain and mutant strain. Data represent the average values and standard deviations from three replicates. Statistical significance was performed using a one-way analysis of variance to compare the CPC concentration differences between A. chrysogenum 1-D1 and each of the two mutants (***p <0.001; **p < 0.01; *p < 0.05).

    图  10  CPC合成与分解相关基因

    Figure  10.  Genes involved in CPC synthesis and decomposition

    图  11  出发菌株与突变株发酵液高效液相色谱图。

    Figure  11.  DCPC content comparison of starting strain and mutant strain. Fig. A: original strain (1-D1); Fig. B: Mutant strain Ac-ΔcahB; Fig. B: Mutant strain ΔcahB::cefG

    图  12  出发菌株与突变株DCPC含量比较。

    Figure  12.  DCPC content comparison of starting strain and mutant strain.

    Data represent the average values and standard deviations from three replicates. Statistical significance was performed using a one-way analysis of variance to compare the DCPC concentration differences between A. chrysogenum 1-D1 and each of the two mutants(***p <0.001; **p<0.01; *p<0.05)

    图  13  DCPC乙酰转移酶基因cefG表达量测定

    Figure  13.  Determination of cefG expression of DCPC acetyltransferase gene

    Data represent the average values and standard deviations from three replicates

    表  1  引物列表

    Table  1.   List of primers

    Primer nameSequence(5’-3’)
    sgcahB-1aactcaccgcgacgtAAGCTTtgccgc
    CTGATGAGTCCGTGAGGAC
    sgcahB-2catgtggccggtcttgccgc
    GACGAGCTTACTCGTTTC
    sgcahB-3gcggcaagaccggccacatg
    GTTTTAGAGCTAGAAATAGC
    sgcahB-4cagtaacgttaagtg
    ACTAGTTCGAGATGACCCAATGTCC
    R-pAN7-1-FttaatgtgaTAAagtagatgccgaccgcgg
    R-pAN7-1-Rgaggcgacatggtgatgtctgctcaagcgg
    pAN-cefG-Fagacatcaccatgtcgcctcagatcgccaatcgct
    pAN-cefG-Rgcatctacttcacattaatgactgatcgaggaatcc
    cahB-B1-F1gatgccatcacgagatttacgaaga
    cahB-B1-R1acaagggaattcccgtatagtctccgtctccataatc
    cahB-B2-F1ctccactcgaggactctccttattcctgcttacac
    cahB-B2-R1gccacttgattcagtaaccttctatg
    cahB-PT-F1agactatacgggaattcccttgtatctctacacacagg
    cahB-PT-R1tgttagtcaagctgcgatgaagt
    cahB-PT-F2gttgacaaggtcgttgcgtc
    cahB-PT-R2gaataaggagagtcctcgagtggagatgtggagtg
    C-veri-Fcgtcgagtacattgagcaggacgccattgt
    C-veri-Raaaggtttgtggaacacacgccagtgtccc
    C1-1gcagcacctacacctatgatgattctgc
    C1-2ttactggccgttgttgatgagaaggt
    C2-1attggaatgaacatgaatctgaggactgca
    C2-2gcttacctaccgaacatcatctcgaatcgt
    C3-1gggtacatactcatagcaacctacattg
    C3-2tacagagtcgaagaatatcctcttgacacc
    C4-1attaatgcattgactgcaacctagtaacgc
    C4-2gaggttaatctcgataacacacccaacaat
    cefG-F1cacgacgattcgagatgatgtt
    cefG-R1ccgactacggcagcaattt
    *大写字母为模板质粒pUC57中RGR特异性结构的骨架
    下载: 导出CSV
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出版历程
  • 收稿日期:  2020-06-23
  • 网络出版日期:  2020-12-16

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