| Aspergillus niger is widely used in the production of commercial proteins,but the genetic manipulation tools for A.niger are relatively simple.The traditional homologous recombination system has the characteristics of simple operation and wide application range,but its efficiency is generally low,so a set of simple and efficient genetic operation tools is urgently needed to be developed.A.niger has a strong ability to secrete extracellular proteins,the expression of heterologous proteins in A.niger is not satisfactory.Analysis of its limiting factors shows that the transcription of heterologous protein genes and the levels of m RNA stability are generally well.Its production bottlenecks mainly exist in the modification process of the post-translational secretory pathway of proteins,such as the defect in protein folding in the endoplasmic reticulum(ER),the ER degradation phenomenon under protein processing pressure,the resource consumption of secretion of a large number of endogenous proteins,and so on.As the main component of ER,phospholipids have an important influence on the morphology,volume and function of ER.It has been reported in Saccharomyces cerevisiae that the transcription factor(TF)ino2 and ino4,which mainly regulates phospholipid metabolism,have a leading role in regulating phospholipid metabolism,which in turn affects the function of the ER,and also affects the homeostasis of the ER protein processing.However,in A.niger,few studies have been conducted on the regulation of phospholipid metabolism.If there is/are TF(s)that regulate phospholipid metabolism in A.niger and how they are regulated is the first scientific question we raised.The imbalance of ER phospholipid homeostasis will cause disturbance of the lipid bilayer composition,which will affect the ER morphology.This change can activate the ER UPR effect independently of the UPR pressure.In addition to serving as a response pathway to ER stress and promoting control of intracellular protein quality control and homeostasis,there are also many functional genes related to lipid metabolism in UPR regulatory genes associated with the protein secretion pathway.There probably have some intrinsic relationship between ER protein homeostasis and phospholipid metabolism homeostasis,but there is currently no clear analysis.If there is a relationship between phospholipid metabolism homeostasis and ER protein homeostasis in A.niger,this is second scientific question we raised.In this thesis,the CRISPR / Cas9 gene editing technology suitable for A.niger was established by discovering the U6 promoter(p An U61 and p An U62)of A.niger and combining the principles of homology-mediated repair and in vitro transcription of sg RNA.Phospholipid synthesis regulatory mechanism was revealed by excavating phospholipid regulating transcription factor ino2 and conducting genetic studies on it.Then the regulation mechanism of phospholipid synthesis was explored by discovering key genes in regulation of phospholipid(ino2,cho2,opi3 and dgk1)and conducting genetic research on it.By modification of the genes in regulation of phospholipid metabolism and UPR effector gene(hac A),phospholipid homeostasis imbalance strains(?ino2,?cho2,?opi3,and ?dgk1)and ER protein homeostasis imbalance strains(?hac A and hac Ai)were constructed.Based on the strains above,the intrinsic relationship between phospholipid homeostasis imbalance and ER protein homeostasis imbalance were investigated.The specific research content are as follows:1.Construction of gene modification based on CRISPR-HDR strategy in A.niger.First found and applied the III type promoters(p An U61 and p An U62,which have applied for an authorized patent)to its own CRISPR system in A.niger.An efficient CRISPR-HDR system was established combining the promoters above with homologous-directed repair and in vitro transcription of sg RNA.Using this CRISPR-HDR system,we successfully deleted two 50 kb of secondary metabolic gene clusters and simultaneously inserted multiple copies of glucose oxidase Gox C at multiple sites to increase its enzyme activity to more than 4 times that of strains transformed based on traditional methods were.The CRISPR-HDR genome editing technology provides an efficient tool for the lab and the construction of the gene-deficient strains below;2.Study of the mechanism of b HLH TF ino2 and its regulation of phospholipid metabolism in A.niger.First identified and characterized the b HLH type TF ino2(An02g04350),then the mechanism of ino2 was determined by EMSA and yeast two-hybrid: it combined with itself to form a homodimer,and combined with the E-box motif(CASSTG)to regulate the transcription of genes related to phospholipid synthesis.Transcriptomics studies of ino2 knockout strains revealed that ino2 is important to regulate genes related to the metabolism of the cell wall,the activity of choline dehydrogenase,fatty acid synthase,lipid metabolism,ammonium ion transport across the membrane and ER related protein metabolism in A.niger.Observation of phenotype plate of ino2 disruptant strain and analysis of different gene sets reveled that ino2 can affect the cell wall and DNA damage repair system.Studies based on HPLC-MS / MS found that the absence of ino2 causes a PI homeostasis imbalance of A.niger.3.Study on the interaction between the phospholipid homeostasis imbalance and ER protein homeostasis imbalance in A.niger.First identified the key genes cho2(An15g06310),opi3(An08g00560),and dgk1(An02g09160),which are critical in synthesis of PE,PC and PA,through bioinformatics and phenotype observation.Based on phospholipid homeostasis imbalance strains(?cho2,?opi3,and ?dgk1)and ER protein homeostasis imbalance strains(?hac A and hac Ai),on the strength of HPLCMS-based lipidomics and transcriptome analysis,we concluded that: Homeostasis imbalance of phospholipid will cause homeostasis imbalance of ER proteins,and homeostasis imbalance of ER proteins will cause homeostasis imbalance of total phospholipids in A.niger. |
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