Establishment Of A Highly Efficient Gene Targeting System In Aspergillus Oryzae Based On The Ku70Gene Marker-free Deletion

The filamentous fungus Aspergillus oryzae （A. oryzae） is a kind of importantindustrial microorganisms widely applied in food processing and fermentationindustry due to its high production of proteases and carbohydrates and its safety forfood production. Unfortunately, the high frequency of nonhomologous end joining（NHEJ） has reduced the gene targeting frequency （mostly lower than10%） andhampered the gene manipulation in A. oryzae. In order to solve this problem, a highlyefficient gene targeting system of a wine production strain A. oryzae C-2wasestablished. To improve gene targeting efficiency of A. oryzae by homologousrecombination, ku70gene, one of the key factor in NHEJ pathway was deletedthrough a marker-free deletion method.1. Conditions for A. oryzae protoplasts formation and regeneration were optimized.The optimal conditions were obtained as follows: the mycelia was cultured for20h,then treated by enzyme mixture containing10mg/mL of snailase and20mg/mL ofcellulase, enzymolysis in a50mL flask with80rpm oscillating at30℃for3h;obtained protoplasts were regenerated in medium containing1.2M sorbital. Underoptimal conditions, the protoplast yields was2.44×10⁷/mL and regeneration rate was51.0%, which makes it possible to establish the highly efficient transformationsystem.2. A ku70maker-free deletant kuD6-3was constructed through the PEG/CaCl2induced protoplast transformation method. Because the bidirectional marker genepyrG was removed according to the marker-free deletion method （Latour system）,kuD6-3was constructed with the recyclable pyrG marker and without any exogenoussequences remaining, it could be quite suitable for further genetic manipulation.3. The xylitol dehydrogenase allele xdh gene was disrupted by insertionalinactivation, the efficiency of homologous integration in the ku70deletion strain forxdh was increased to73.3%compared with the4%in the original strain C-2.Meanwhile, no irregularities in growth rate, vegetative growth, mycelial morphology,spore formation and germination were observed in kuD6-3. Sensitivities to DNA damaging agents including UV, hygromycin B, methyl methane sulfonate （MMS） andH₂O₂were not significantly different between kuD6-3and C-2. In all cases, kuD6-3showed no sensitivity differences to osmotic pressure in comparison with C-2. Theseresults indicated the absence of ku70could significantly improve gene targetingefficiency in A. oryzae without changing its growth phenotypes or weakening itsability to heal damage to itself. Therefore, kuD6-3will be an excellent host formolecular breeding.