The Construction Of Molecular Operation Platform In Acremonium Chrysogenum And Exploration On The Function Of Sorbicillinoids Biosynthetic Genes

The market of beta-lactam antibiotics has tremendously increased in the global market.Due to the broad-spectrum antimicrobial property and low toxicity,the demand for cephalosporin C(CPC),which is the precursor of Cephalsporins,is growing continuously.Acremonium chrysogenum,as the main industrial strain of CPC production,has been studied extensively in the field of fermentation process and molecular modification.However,the CPC titer is far much lower than that of penicillin.It is reasonable to believe that on the basis of traditional methods,significant improvement of CPC production can be achieved with the genetic engineering application.To date,however,the techniques for genetic engineering of A.chrysogenum are challenging and the transformation efficiency is extremely low because of the complicated cell wall structure and rare conidiospore production as well as the special sexual life cycle.The phenotype of wild A.chrysogenum is yellow,and two PKS,SorA and SorB,have been reported to be supposed to synthesize the basic hexaketide scaffold and were both indispensable in yellow pigment production.As a proof of principle,the two genes were chosen as the targets of genetic modification.The main propose of this work was to establish the molecular operation platform of A.chrysogenum,and subsequently,to investigate the regulation mechanism of related genes in yellow pigment(sorbicillinoids)synthesis.Firstly,the Agrobacterium-mediated transformation platform was successfully constructed.A.chrysogenum CGMCC 3.3795 was employed for transformation,and hygromycin,pAgl-H3 and supervirulent AGL-1 were chosen for the selection marker,the vector and the infect bacteria,respectively.The plasmid containing both sorA and sorB homologous arms was constructed and transformed into A.chrysogenum,and A.chrysogenum-?pks strain which knocked sorA and sorB out simultaneously was successfully constructed.The mutant could not produce the yellow pigment and the fermentation results showed the CPC production of mutation strain was improved obviously.The efficiency of homologous recombination in this established platform was about 10-20%.Secondly,the genetic engineering platform based on CRISPR/Cas9 technology was successfully constructed.The constructed template plasmid consisted of three components,i.e.Cas9 expression cassette,sgRNA expression cassette,and the hph selection marker.The knockout plasmid was transformed into A.chrysogenum protoplasts through the traditional PEG mediated transformation method.Consequently,two mutation strains were screened out,respectively,as A.chrysogenum-?sorA which lacked SorA and A.chrysogenum-?sorB which lacked SorB.The efficiency of CRISPR-Cas9 mediated gene editing established in this study was very high,which reached over 80%.Finally,through the sorbicillinoids biosynthetic gene cluster expression profile analysis in A.chrysogenum-?sorA and A.chrysogenum-?sorB,we found the deficiency of SorA and SorB would decrease the expression of sorbicillinoids synthesis-related genes,and then hinder the synthesis of sorbicillinoids.The results indicated that to some extent,sorbicillinoids biosynthetic gene cluster was regulated through an autoinduction mechanism and the cluster expression profile was influenced by the products of its own synthesis pathway.