Efficient Expression Chassis Construction And Expression Element Optimization In Penicillium

Filamentous fungi are widely used in industrial production as a homologous and heterologous protein expression hosts,which possess a series of advantages such as low requirements of raw material,excellentpost-transcriptional modification,and strong secretion ability.However,it is reported that filamentous fungal protein expression systems still have some shortcomings such as high secretion background or low target protein expression efficiency.Penicillium oxalicum is a lignocellulose-degrading enzyme-producing fungus with independent intellectual property rights in China,it has high secretion ability and low extracellular protein background when using starch as the sole carbon source for cultivation.In this study,the secretory background of the host was reduced by knocking out the Amy13 A protein and utilizing the non-inducer starch as a carbon source.The strong promoter Pamy15 A was further improved by overexpressing the transcription activator AmyR and deleting of putative depressor CreA.The main research contents and results of this article are as follows:1.Deletion of Amy13 A reduced the extracellular protein background and made the native amylase Amy15 A a reporter.The amy13 A deletion cassette was constructed by using homologous flanking of amy13 A and pyrG gene from Aspergillus nidulans as a selection marker.The strain Δ13A was obtained after transforming the parent strainDB2 with amy13 A deletion cassette.Then,we observed the Δ13A with a reduced secretion background and a higher efficiency of amy15 A promoter when cultured on starch compared with that on glucose.2.Further enhancement of Pamy15 A transcription by overexpression of AmyR with the amy13 A promoter.The overexpression of transcriptional activators can significantly increase the expression efficiency of downstream regulated genes.Therefore,the stratagy that using the coding region of amyR to replacethe amy13 A coding region were performed in the parent strain DB2.As a result,the engineered strain Δ13A-OamyR was obtained and its efficiency of the promoter Pamy15 A was improved after overexpressing of AmyR and cultivating on starch medium.3.Deletion of CreA dramatically increased Pamy15 A transcription levels.The CreA knockout cassette was constructed by double-joint PCR and introduced into the parent strain Δ13A-OamyR after eliminating its pyrG gene by a β-rec/six self-excising marker recycling system.The transformant Δ13A-OamyR-ΔCreA was obtained and showed a significantly increase in amylase activity and extracellular protein concentration when using starch as the sole carbon source.Meanwhile,the trnascription efficiency of amyR and the promoter Pamy15 A were further enhanced.