The Construction Of The Related Tools Used For Cephalosporium Acremonium Genetically Engineering

Cephalosporins are a family of theβ-lactam antibiotic which kill bacteria by interference with the synthesis of bacterial cell wall and destruction of cell wall.Due to wide arrange,higher activity and lower toxicity of antibiotics, Cephalosporins have been regarded as the major antibiotics together with Penicillin,since the industrial production during 60^th of 20 century.In the present study,vgb gene was integrated to the chromosome of Cephalosporium acremonium.By suitable promoter,vgb gene was constitutively expressed to improve the transport of oxygen and the growth of Cephalosporium acremonium under hypoxygen.Therefore,the concentration and quality of Cephalosporium acremonium were improved,the cost of fermentation was decreased,and the yield of Cephalosporin C was enhanced, providing new way to the contradiction of the need of oxygen and the limited provision of oxygen by the instruction during the fermentation of Cephalosporium acremonium. The construction of the transform system of Cephalosporin was necessary for the study of the molecular biology of Cephalosporium acremonium.An efficient tansformation vector was determined by the strength of the promoter of expressing genes.To express extra gene,the promoter should be recognized by the RNA polymerase of Cephalosporium acremonium.Therefore,to construct a transformation vector,the clone of proper sequence of promoter is required.In the present paper,the promoter pcpC_p of IPNS and pcpC(452bp) promoter of IPNS was cloned.By enzyme digestion and sequencing,the cloned promoters were identical to the data in NCBI.The cloned promoters can be recognized by the RNA polymerase of Cephalosporium acremonium and can initiate the expression of extra gene in Cephalosporium acremonium.In this paper,in order to obtain high yield and purity VHb protein used for antibody production and ELISA,we contructed plasmid pET28a-vgb.We found the second amino acids gene code have the determinants affect of VHb expression.VHb protein can be purified through metal affinity chromatography by His-Tag label structure.In order to clone and express vgb in Cephalosporium acremonium,we have studied the preparation and regeneration conditions of Cephalosporium acremonium protoplast.We have identified the condition of medium,culture condition,enzyme,and Osmotic stabilizer.Used 1000unit·mL^-1 Lywellzyme as enzyme,0.6 mol·L^-1 NaCl solutions as Osmotic stabilizer,the protoplast number can be 5.86×10⁶·mL^-1,Regeneration rate can be 41%. To integrated vgb gene in the chromosome of Cephalosporium acremonium,we constructed the transformation vector of Cephalosporium acremonium,i.e.pDH25-pcpC-vgb.This plasmid can transform Cephalosporium acremonium and integrate to the chromosome of Cephalosporium acremonium with HygB as selection marker,the RNA polymerase of Cephalosporium acremonium can recognize promoter pcpC and promoter PtrpC,and separately initiate the expression of vgb gene and Hyg B resistance gene.vgb both used TtrpC termination sequence,providing basis for the transformation of Cephalosporium acremonium.In this study,we have made a good achievement in the genetic engineering of Cephalosporium acremonium,and the research can also afford the foundation of vgb clone and expression in Cephalosporium acremonium.The results are also beneficial to reconstruct other antibiotic-production fungus.