Engineering An Industrial Acremonium Chrysogenum To Improve The Production Of Cephalosporin C And Reduce The DCPC Content Using CRISPR/Cas9 System

Cephalosporin C(CPC)is a class of secondary metabolites produced by the filamentous fungus Acremonium chrysogenum,whose semisynthetic derivatives play an important role in the pharmaceutical industry owing to its efficacy and fewer side effects.In the fermentation production of A.chrysogenum,there are some intermediate products such as deacetylcephalosporin C(DCPC)remaining in the broth as impurities,which affects the yield of CPC and adds difficulties in separation and purification.However,due to the limited fermentation capacity of A.chrysogenum,the demand for CPC still far exceeds its supply.Therefore,how to increase the production of CPC has become an urgent problem.At present,the synthetic pathway of cephalosporin C has been clearly clarified and it is evidenced that CPC-acetyl-hydrolase exists in the cell.It is also well known that morphogenesis is closely linked to secondary metabolism but we know little about the regulatory mechanism.The CRISPR-Cas9 mediated gene editing makes gene modification easier and more efficient,which provides a good platform for improving both production and quality of CPC and exploring its regulatory mechanism.Here,we researched about CPC-acetyl-hydrolase gene cahB and the type I integral plasma membrane protein Axl2(Axial budding pattern protein 2),a central component of the bud site selection system(BSSS)using the CRISPR-Cas9 mediated gene editing in a CPC industrial producer 1-D1.In this study,we constructed a higher-yield CPC producer and carried out a preliminary investigation of regulatory mechanism between morphological changes and production of CPC.Firstly,we used the CRISPR/Cas9 system to knock out the cahB to reduce the accumulation of impurities DCPC,and obtained a strain Ac-ΔcahB with2 bp deletion.Secondly,we inserted donor DNA which contained a cefG gene that encodes DCPC acetyltransferase into the cahB locus by using the HDR and CRISPR,and thus the cahB deleted strain combined with cefG overexpression capability Ac-ΔcahB::cefG,was obtained.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,which increased by approximately 32.6%compared to the control strain(1D1).Moreover,for the determination of DCPC content,the impurity of CPC production,the ratio of the peak area of DCPC to CPC was employed.The results showed that the DCPC content of the original strain was 12.56%,while that of strain Ac-ΔcahB decreased slightly with the content of 11.33%of the DCPC content.However,in the strain Ac-ΔcahB::cefG was dramtically dropped to 6.81%,which decreased significantly(P=0.001797)compared to the control strain.The results suggested that the overexpression of cefG gene by the strong promoter gpdA greatly increased the activity of acetyltransferase,thus promoted the conversion of DCPC to CPC,significantly reduced the accumulation of DCPC and improved the production of CPC.In the aspect of mycelium morphology,we studied the Acaxl2(Axial budding pattern protein 2),which is the key component in the bud site selection system(BSSS).Using the CRISPR/Cas9 system and HDR,we inserted egfp donor DNA into the Acaxl2 locus,generating the strain Ac-AAcaxl2::eGFP.The CPC titer of the mutant strain was 5573 μg/ml,which is nearly three times higher than that of the parental strain 1-D1.The mycelial morphology of the mutant strain Ac-ΔAcaxl2::eGFP was mainly swollen hyphal morphology without separation,while that of the control was mostly existed as isolated big arthrospores.The amount of arthrospores of the mutant strains in the middle and late fermentation reached 90%,and the control strain decreased from 95%at 48 h to 54%at 168 h.Furthermore,we found that the expression of global regulator Acfkhl and cpcrl was slightly downregulated at the transcriptional level at fermentation time 72 h and 96 h,respectiviely.However,the expression of the essential CPC synthetic genes(pcbAB,cefEF,cefG)were up-regulated by 1.89,18.55 and 2.49 times respectively at fermentation time 72 h,and were increased by 3.19,8.19 and 1.79 times at 96 h respectively.Among them,cefEF had the largest increase.Moreover,the expressions of Acbud3 and Acbud4 also increased slightly in the BSSS pathway.The results suggested that the deleation of Acaxl2 accelerates the formation of arthrospore,leading to the remarkerable increase of the yield of CPC.