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    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), 2021, 47(4): 427-437. 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), 2021, 47(4): 427-437. DOI: 10.14135/j.cnki.1006-3080.20200623003
    XU Yan, FENG Tao, 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, 2021, 47(4): 427-437. DOI: 10.14135/j.cnki.1006-3080.20200623003
    Citation: XU Yan, FENG Tao, 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, 2021, 47(4): 427-437. 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

    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效价没有提高但杂质含量略有下降,而Ac-ΔcahB::cefG不仅产量比对照提高约32.6%,达6 072 μg/mL,而且DCPC杂质含量(DCPC峰面积/CPC峰面积)显著降低到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。

       

      Abstract: The CRISPR/Cas9 mediated gene editing was used in a cephalosporin C (CPC) industrial producer Acremonium chrysogenum 1-D1 to improve the production and quality of CPC by reducing the accumulation of by-product deacetylcephalosporin C (DCPC). Firstly, we used the CRISPR/Cas9 system to knock out the CPC acetylhydrolase gene cahB to reduce the accumulation of impurities DCPC, and a strain Ac-ΔcahB with 2 bp deletions in cahB was obtained. Secondly, we inserted donor DNA containing a cefG gene, which encoded DCPC acetyltransferase into the cahB locus by using the HDR and CRISPR, thereby resulting in the cahB deletion strain Ac-ΔcahB::cefG combined with cefG overexpression capability. It was found that the CPC production of strain Ac-ΔcahB was increased slightly, while strain Ac-ΔcahB::cefG produced 6072 μg/mL of CPC after 168 h cultivation, which was increased by 32.6% compared to the original strain (1-D1). Moreover, the ratio of the peak area of DCPC to CPC was employed to determine the DCPC content. The results showed that the DCPC content of strain Ac-ΔcahB was slightly decreased from 12.56% to 11.33% of original strain. However, the DCPC content in the strain Ac-ΔcahB::cefG was dramatically dropped to 6.81%, which decreased significantly (p=0.001797) in comparison to the original strain. In addition, the expression levels of the acetyltransferase gene cefG in both functional strains were increased in 72 h and 96 h, respectively, and the transcription level of Ac-ΔcahB::cefG strain was increased by 5 times at 96 h. These results suggested that the overexpression of cefG gene by the strong promoter gpdA greatly increased the transcription of acetyltransferase gene, and promoted the conversion of DCPC to CPC. Thus, the accumulation of DCPC was significantly reduced and the production of CPC was improved. The CRISPR/Cas9 system combined with donor DNA is a more efficient gene editing method to be applied to industrial Acremonium chrysogenum.

       

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