Construction And Optimization Of A Novel Clean-background Expression System In Penicillium

Recombinant expression of protein has been widely used in life science,biotechnology,medicine,and materials science,which has created great social benefits and economic value.In the process of expression of recombinant protein,an appropriate expression system can be selected according to the protein properties to improve its expression efficiency.Currently,there are many protein expression systems.Among them,the filamentous fungus expression system has got wide attention due to its low raw material requirements,good post-transcriptional modification,and high secretion ability.Many filamentous fungi of different species have been developed into protein expression host strain.Penicillium oxalicum is a lignocellulose-degrading enzyme-producing strain with independent intellectual property rights in China,which has the characteristics of excellent protein secretion ability and natural low protein secretion background under non-induced conditions.Some research is carried out for the construction of a low-background expression chassis from Penicillium oxalicum based on its good genomics and proteomics research.Furthermore,a mature and universal protein expression platform for filamentous fungal protein expression was constructing by the screening of high-efficiency expression elements,high-purity expression,and optimization of recombinant proteins,etc.The main research contents and results of this article are as follows:1.Construction low secretion chassis cells from Penicillium oxalicumThe amy15 A deletion cassette was constructed by using homologous flanking of amy15 A and the pyr G gene as a selection marker.The strain?13A15A-Oamy R was obtained after transforming the engineering strain?13A-Oamy R which was constructed by our previous study with an amy13 A deletion cassette.The extracellular protein background was further decreased by knocking out amylase Amy15 A in?13A15A-Oamy R.2.Screening strong promoters to improve the expression of recombinant proteinTwo strong promoters were selected in this study.One is the promoter Pamy15A(inducible)of the amylase Amy15 A that is further highly expressed in the strain ?13A-Oamy R due to the overexpression of Amy R,and the other is the strongest constitutive promoter Pubi D in Penicillium oxalicum that has been reported in the previous study.3.Identification of core promoter of strong promoter Pamy15APamy15A is a highly efficient and inducible promoter found in Penicillium oxalicum when cultured with starch as the sole carbon source.We used the method of promoter gradient knockout to obtain transformants with the promoter region length of Pamy15 A ranging from300 to 1400 bp(with an interval of about 100 bp).The promoter efficiency was compared by measuring the activity of the amylase Amy15 A in the transformants.The core elements of Pamy15 A were also identified and analyzed.4.Selection of stronger promoters and continuously activation of G protein-c AMP to further increase recombinant protein expressionThe RNA-Seq technique was used to analyze the highly expressed genes in the ?13A-Oamy R strain,and it was found that the PDE?07911(Extracellular membrane protein,Emp A)gene was the maximum expressed.Therefore,its promoter(referred to as Pemp A)was selected to express a recombinant protein in the ?13A15A-Oamy R strain.By comparing with the protein expression efficiency under the control of the promoter Pamy15 A,it is indicated that in the ?13A15A-Oamy R strain,the promoter Pemp A can improve the expression of recombinant protein.By continuously activating the G protein-c AMP pathway in recombinant protein expression engineering strain,the expression of recombinant protein can be further specifically improved.5.Exploratory study on heterologous expression of peptide in?13A15A-Oamy R strainUse one-step cloning to introduce the codon-optimized target gene into the universal protein expression plasmid h-Pemp A-T15 A which was under the control of the promoter Pemp A.Then,the constructed amylase Amy15 A and Amy13 A double deletion strain ?13A15A-Oamy R was used for heterologous expression of the target polypeptide.