Expression Of Acidic Protease Gene In Pichia Pastoris And Aspergillus Niger

Acidic protease is an enzyme which can hydrolyze proteins in the acidic environment, and the optimum pH is from 2.5 to 5.0.As has good acid resistance, Acidic protease is widely used in food, medicine, light industry, leather technology and forage processing industry. Aspergillus usamii is the main species in the industrial production of acid protease, but the expression is regulated by the deter at cabon and nitrogen, induced by proteins, and controlled by the pH. The result is that it is difficult to control the ferment condition and the activity is unstable.In addition, the activity of acid protease produced in Aspergillus usamii needs to be further improved.Eukaryotic expression system is an effective method in the efficient expression of proteins. Yeast as an engineering strain has received more and mor attention. It has the functions that regulates gene expression and modifies proteins. Meanwhile it overcomes the questions of low activity, denaturation and renaturation of Inclusion body, and so on. Moreover, it could be high-density fermented and the product is similar to natural proteins. The clinical application is safe. Aspergillus niger has been more and more attractive because of the excellent expression and secretion ability. It has the eukaryotic secretion mechanism and may also has the protein modification performance, such as high mannose-type and N-glycosylation. It is an ideal expression system for eukaryotic gene. Efficient expression of acidic protease in yeast and Aspergillus niger by means of genetic engineering has important significance in simplifing the fermentation conditions and increasing the production.In this study, cloned pepA gene with gene donor Aspergillus usamii, then constructed yeast expression vector, transformated into Pichia pastoris by electroporation, and got engineering strain that express acidic protease gene effectively. Meanwhile, tried to obtain Aspergillus niger as the acceptor with the pyrA gene and kusA gene disrupted mediated by Agrobacterium, and constructed homologous recombination vector of pepA. Laied the basis for establishing the Aspergillus niger expression system and achieving the efficient expression of pepA gene in Aspergillus niger. The major results were as follows:1. Cloning of genesThe cDNA sequence of pepA gene was cloned from Aspergillus usamii by RT-PCR. PyrA gene, 5' and 3' homologous arm of ku70 gene, promoter and terminator of glaA gene were cloned from Aspergillus niger by PCR. PyrG and pepA genes were cloned from Trichoderma reesei and Aspergillus usamii respectively using PCR. The sequences of these genes were right.2. The expression of pepA gene in Pichia pastoris Pichia pastoris expression vector pGAPHαm-pepAc was constructed. The linear expression vectors were transformed into Pichia pastoris by electroporation. Transformants were identified by PCR: GS115-pGAPHαm-pepAc. Under optimum fermentative condition, the activity of the acid protease expression was 1289.68u/mL.3. Construction of Aspergillus niger expression vectorsFive Aspergillus niger expression vectors were constructed, incuding pEMT-pyrA,pEMT-△pyrA,pEMT-△kusA,pEMT-△kusA-pyrG,pEMT-△glaA–pepA,and then were translated into Agrobacterium GV3101 for Aspergillus niger transformation.4. Construction of the pyrA gene disruption mutant in Aspergillus nigerThe expression vector pEMT-△pyrA was transferred into Aspergillus niger UV48 mediated by Agrobacterium Tumefaciens. At the stage of screening, 5-FOA was added in screening medium.Some resistant mutants were obtained via screening. However, these were not pyrA gene disruption mutants by PCR identified. The study is still in research.