Construction of CPC High-Yield Industrial Strain with Low DCPC Content Using CRISPR / Cas9 System

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.