Construction Of Gene Disruption Mutants In Aspergillus Niger And Functional Analysis Of The Genes

Aspergillus niger is one of the most important microorganisms widely used in a variety of industrial processes including the production of a broad spectrum of extracellular enzymes [glucoamylase, (hemi-)cellulase, and phytase] and organic acids such as citric acid. More recently, this fungi has been developed as a host organism for the production of foreign (heterologous) proteins, especially for the production of recombinant proteins supplied for food, environmental and pharmaceutical industries. The exploration of Aspergillus niger was considered to be very attractive as it has a number of advantages compared with other expression systems. These advantages include the high efficient capacity of protein secretion, post-translational modifications such as glycosylation and disulfide bridge formation closer to high eukaryotic organisms than bacteria and yeasts, optimized fermentation technology, GRAS (Generally Recognized as Safe) approval in production process and so on. Although several proteins have been successfully expressed in Aspergillus niger, the secreted yields of many heterologous proteins, especially mammalian proteins, rarely reach the gram-per-litre level. One of the major obstacles in achieving high level protein expression and secretion is the proteolytic degradetion of the expressed heterologous protein by host proteases, as heterologous proteins are often more prone to proteolysis than homologous proteins. Bioprocessing steps such as downstream processing at low temperatures, the improvement of pH, the early separation of product and protease(s) or the use of protease inhibitors might reduce proteolysis. However, they will certainly not eliminate the problem because much of the degradation occurs in vivo during the production of the protein. Disruption or deletion of the proteases of the host is supposed to be a very powerful approach to reduce proteolytic degradation of the expressed products and to improve protein production.In this thesis, two A. niger putative secreted protease genes, the pepD and the pepAd genes, and one A. niger gene involved in the secretory protein degradation process in endoplasmic reticulum (ER), the derl gene, were disrupted using homologous recombination technology in order toimprove heterologous protein expression and secretion in Aspergillus niger. In the resultant strains, ApepD, ApepAd and Aderl, activities of the target heterologous protein laccase were tested respectively to study the influence of disruption of these genes on the degradation of heterologous protein in A. niger.Main results of this research are shown as follows:1. The pepD gene fragment was amplified by PCR with Aspergillus niger GICC2773 genomic DNA as template and cloned into vector pBS. The selection marker, a hygromycin resistance (hph) expression cassette, was inserted into the pepD gene resulted in the disrupted plasmid pBSDH. The plasmid was completely digested with Stu I to generate a 3.7kb linear fragment, in which the hygromycin resistance expression cassette was flanked with a 1067bp 5' fragment and a 1180bp 3' fragment of the pepD gene. It was then used to transform GICC2773 strain by PEG-mediated transformation. The recipient GICC2773 contains an integrated laccase expression cassette. Total 84 transformants were obtained by hygromycin resistance selection, and were screened by PCR using a primer annealing to the hph gene and a primer annealing to the sequences outside the 3' homologous fragment. Transformant A pepD66 was identified to be apepD gene disruption strain. The effect of pepD disruption in A pepD66 on laccase expression was analysed. The result indicated that the disruption of pepD gene improved heterologous protein laccase production by 6-7% without any negative effect on the total protein production. Disruption of the pepD gene did not have any negative effect on growth. Thus disruption of the pepD gene in a recipient strain would be helpful for protecting the heterologous proteins from degradation.2. The recombinant plasmid pBSAdH for the pepAd gene disruption in Aspergillus niger was constructed. The plasmid contained the pepAd gene upstream (P) 1213bp and downstream (T) 943bp homologous fragments with insertion of the expression unit of the hygromycin resistance gene (hph) between them. The P and T DNA fragments were synthesized by PCR from Aspergillus niger genomic DNA. The plasmid was completely digested with the Hpa I. The resultant 3.7kb linear fragment was introduced into the Aspergillus niger GICC2773 containing laccase expression cassette by PEG-mediated transformation. 89 hygromycin resistance transformants were screened, and one strain named A pepAdl9 was identified to be the pepAd gene disruptant by PCR analysis. Data of functional assay of the A pepAdl9 strain indicated that heterologous protein laccase production was hardly improved.3. The derl gene disruption plasmid pMW-derlH was constructed, in which the selection marker hygromycin resistance (hph) expression cassette was flanked with 831bp 5' homologous fragment and 998bp 3' homologous fragment. The disruption plasmid was completely digested with Hpa I and Nru I and the resultant mixture of linear fragments was then used to transform GICC2773...