Development Of A Self-cloning System For Aspergillus Oryzae3.951in The Soy Sauce Industry

Aspergillus oryzae, a widely used filamentous fungus in traditional fermentation of foods, paeticularly important in the soy sauce industry, can produce amounts of proteases and one of safety microbial strain announced by US Food And Drug Administration, as an expression of homologous of homologous and heterologous proteins excellent. Utilization ration of raw material and amino acid yield rate had a direct impact on the costs of raw materials and market competitiveness. Therefore, the development of A. oryzae with high protease activity possess very important significance. In this study, using an important strain A.oryzae3.951from soy sauce brewing industry in China as the original strain, a homologous transformation system based on food-grade and the producing neutral protease self-cloning engineering A. oryzae. The main contents are as follows:1The distribution of nuclei in each conidium for A. oryzae3.951.The optimized conditions of DAPI staining were5%sodium hypochlorite treatment conidium wall for5min,8μg/mL DAPI for2h. the spores within the nucleus that glows bright blue fluorescence.contained1-4nuclei in each conidium through DAPI staining conditions optimized. The percentages of uninucleate and binucleate conidia were approximately16.15%and74.22%, respectively. Most of the uninucleate conidia were distributed from2μm to5μm, with maximum frequency of about3μm. The percentages of uninucleate conidia that passed through5-μm and3-μm membranes increased to51.24%and80.41%, respectively. Although it can not completely remove the multinucleate spores, but significantly increased the probability of a single nuclear conidium.2The development of pyrG mutant for A.oryzae A.oryzae3.951conidia suspension before UV mutagenesis was filtered using a5-μm membrane to remove excess multinucleate conidia. Uninucleate conidia of5-FO A-resistant colonies were enriched by filtration using a3-μm membrane. Thus, the uridine auxotroph mutant strain was obtained and designated as AS11. The genomic DNA of AS11was used as a template to amplify the pyrG gene for sequencing, The results from sequence analysis revealed that there were six point mutations. One of these mutations was due to the frameshift for base insertion, and caused the inactivation of orotidine-5’-monophosphate decarboxylase (OMPdecase) function. From the complementation of A. oryzae AS11, transformants of A. oryzae AS11were restored using the pyrG+phenotype. Compared with A. oryzae3.951, spore production ability was no significant difference, the activity of neutral protease increased36.30%. GFP was used as a genetic reporter, and GFP-pyrG was successfully transformed into AS11using the PEG-CaCl2transformation method.3Construction of self-cloning neutral protease Aspergillus.The activity of neutral protease was increased by two strategies. Expression vectors contained neutral protease expression cassette and replaced gpdA promoter transformated into A.oryzae AS11, transformations were obtain throught pyrG+and niaD-double selection marker. A.oryzae niaD mutants were screended by filtration using a3-μm membrane. Neutral protease activity of gene-engineering strain PN18over the3.951increased50.88%, over the AS11increased10.70%.