Construction Of A Recombinant Protein Expression System In Neurospora Crassa

The advantage of filamentous fungi as industrial production host is based on their natural ability to secret large amounts of proteins.With the advances in fungal molecular genetics and genomics,filamentous fungi are developed as microbial cell factories for the production of recombinant proteins.Due to genetic diversity of filamentous fungi,no single host strain is completely suitable for the production of all the secreted proteins.Recently,there have been reports of the utility of new filamentous fungal system in the production of industrial proteins.Neurospora crassa as powerful model filamentous fungal system,which has been charaterized biochemically and genetically,has been a promising candidate host strain.Several recent studies have used N.crassa as a host for production of recombinant proteins,including plant proteins,vaccines and endoglucanase,indicating that this fungal can potentially be exploited as an expression system.Using functional genomics approaches and full genome deletion strains sets of N.crassa,this study explored the potential of this model filamentous fungal as a recombinant protein expression system.Firstly,this study developed a cellobiose induction system with sextuple gene disruptant LQ-1(?ghl-1 ?gh3-3 ?gh3-4 ?cbh-1 ?cbh-2 ?his-3)as the host strain.Using this system,target proteins driven by various cellobiose-induced promoters-could be expressed very efficiently with low background protein secretion.Meanwhile,this study then constructed expression vectors driven by cbh-1 cbh-2,ccg-1 or tef-1 promoters that produced expressed proteins with C-terminal tev-6xhis-gfp tags to allow us to screen for positive transformants and purify recombinant proteins.Two endogenous enzymes(GH3-4 and CBH-1)were chosen as test proteins for recombinant expression.Western blotting and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis indicated that the recombinant proteins were successfully expressed,with secreted levels reaching as high as approximately 2.77 or 2.83 mg/L.The highest enzyme activities and protein contents were detected in the transformant with the cbh-1 promoter,suggesting that pcbhl was the best promoter for recombinant protein expression in this study.Using the two cellulases as the proof-of-principle targets,this study successfully constructed a system for recombinant protein production in N.crassa under cellobiose induction.With further optimization,this system can be used for industrial protein expression.