| Aspergillus niger is widely used as a natural cell factory for the production of various organic acids,enzymes,and recombinant proteins due to its unique growth morphology and excellent secretion ability.However,compared with the high expression level of homologous proteins,the yield of heterologous proteins produced by A.niger is relatively low.Currently,the analysis of limiting factors indicates that the protein secretion pathway may be the bottleneck of heterologous protein production.In the secretory pathway,the protein quality control mechanism of the endoplasmic reticulum is the key node of protein secretion.When a large number of proteins are poured into the expression or the stress of environmental drugs,the newly formed proteins will be incompletely folded or misfolded,triggering the unfolded protein response(UPR)or ER stress.Under stress induction,its core transcription factor,Hac A,is activated to regulate the expression of a series of target genes related to the protein secretion pathway,thereby maintaining cellular homeostasis.However,studies on the regulatory mechanism of Hac A transcription factors in filamentous fungi are limited to transcriptome analysis,and the binding mechanism between Hac A transcription factors and target gene promoters and its overall regulatory role on the protein secretion pathway have not been deeply resolved.The inefficient transport of heterologous proteins between ER and Golgi is thought to be one of the causes of ER stress.Excellent protein secretion is closely related to the efficient and orderly vesicle sorting and packaging mechanism of the host,especially the receptormediated selective transport of the ER to the Golgi apparatus.How cargo proteins are selectively captured to transport vesicles at the endoplasmic reticulum exit sites(ERESs)is a key scientific problem in the study of the vesicle transport mechanism.The role of vesicle transport receptors has been well characterized in mammals and saccharomyces cerevisiae,but the function of these proteins in A.niger has not been studied.In this study,the A.niger CBS513.88 strain was selected as the main object.By developing a buffer suitable for the osmotic pressure of A.niger protoplasts,CUT & TAG technology was successfully applied to the study of Aspergillus niger transcription factors.By integrating ATAC-seq and DAP-seq methods,an antibody-independent technique for detecting global binding sites of transcription factors in vitro was developed.Using these two techniques,the regulatory mechanism of UPR core transcription factor Hac A under endoplasmic reticulum stress was explored,and the genes of the protein secretion pathway that it targets for regulation were clustered and analyzed.In addition,all functional genes of the endoplasmic reticulum to Golgi vesicle transport system and its cargo transport receptors on the genome of A.niger were identified for the first time,and the effects of each receptor on growth and protein secretion of A.niger were investigated by gene knockdown and overexpression.A high-throughput yeast two-hybrid screening method was constructed to identify the interacting proteome of each receptor and to study the potential transport protein profile of cargo transport receptors.Ani Erv14 receptor,the most significant vesicular transport receptor in A.niger,and its specific transmembrane transport protein function were studied.The specific research content and conclusions of this paper are as follows:(1)Development of global detection techniques for transcription factor binding sites of A.niger and its application in studies on regulatory mechanisms of Hac A,the core transcription factor for quality control of endoplasmic reticulum proteinIt was found that the key to successfully applying the CUT&TAG technique in A.niger is to provide suitable osmotic pressure to maintain protoplast morphology.The CUT&TAG technique was successfully applied to A.niger by optimizing the reagent of the CUT&TAG kit.In addition,an antibody-independent in vitro assay,DAP＿ATAC,was successfully developed by integrating DAP-seq and ATAC-seq.By integrating DAP＿ATAC and transcriptome data,the expression levels of target genes potentially regulated by Hac A were explored.Functional clustering analysis was performed on functional genes related to protein secretion pathways,especially those related to vesicular transport systems.(2)Identification and functional characterization of vesicle transport receptors in A.nigerBased on comparative genomics and phylogenetic tree analysis,the vesicle transport system and protein transport receptor genes of A.niger were excavated.In particular,the gene information of Sec12,a key component of the vesicle transport system,on the genome of A.niger model strain CBS513.88 was complemented.All potential endoplasmic reticulum to Golgi vesicle transport receptors on the A.niger genome were identified and characterized.It was found that the knockout of various receptors had different degrees of influence on the growth of A.niger,among which the knockout of Ani Erv14 had the greatest influence,which seriously delayed the spore germination and mycelium growth rate,and made the mycelium almost lose the tolerance to environmental pressure.Compared with the control strains,the total extracellular protein and glycosylase activity of most of the knockout strains were decreased,but knockout of Ani Vip36 not only had little effect on protein secretion but also increased the glycosylase activity by 10%.Overexpression of each receptor did not significantly change the morphology of the cell,and the improvement of the overall protein secretion level was limited.Only overexpression of Ani Erv14 could effectively promote protein secretion and glycosylase activity.Nine potential receptors in the A.niger genome that assist in the transport of endoplasmic reticulum to Golgi vesicles were identified by homologous alignment.Using CRISPR/Cas9 technology,knockout and overexpression strains of each receptor were successfully constructed,and their basic growth phenotypes,tolerance to environmental stress,and protein secretion levels during fermentation were characterized in detail.(3)To construct a high-throughput protein interaction screening method and apply it to identify the interacting proteome of vesicular transport receptors in A.niger,and to study its potential cargo transport profileCombined with yeast two-hybridization and second-generation sequencing technology,a high-throughput screening method for protein-protein interaction was developed,and experimental procedures such as transformant selection,background library setting,and optimized processing of second-generation sequencing data identified by sequencing were optimized to reduce false positive rates.Using this method,the interacting proteome of six vesicle transport receptors in A.niger was identified.Cluster analysis and point-to-point hybridization showed that Ani Emp47 is preferentially bound to secreted proteins,while Ani Erv14 is specifically bound to transporters.(4)Functional characterization of Ani Erv14 receptor of A.niger and detection of specific cargo proteinsThis study focused on the Ani Erv14 receptor,a vesicular transport receptor with the most significant effect on growth and protein secretion in A.niger,and its transmembrane transporters.It was found that the absence of Ani Erv14 would lead to incomplete conidial pores and slow spore germination,which would lead to the slow growth of mycelium and indirectly affect the accumulation of secreted proteins in the fermentation process.The membrane proteome of the Ani Erv14 deletion strain was determined and 200 transmembrane proteins were found to be significantly decreased,including transporters of various sugars,amino acids,and ions.By fluorescence localization of two monosaccharide transporters,their transport was determined to be related to the Ani Erv14. |
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