Abstract:Aspergillus niger TNA09 is a citric acid producing strain used in industry.Due to a requirement for multiple physical and chemical mutagenesis steps and subsequent screening，it is very difficult to carry out traditional genetic modifications in this organism.In this test，the protoplast transformation method was used on Aspergillus niger TNA09 to successfully construct a strain that will permit ready genetic engineering of this industrial strain.Experimentally determined optimal conditions for protoplast preparation and regeneration were as follows:Fresh spores were grown on CM medium for 5 d，then 1×108Aspergillus niger spores were inoculated into about 100 mL of potato dextrose broth（PDB）liquid medium，and cultured for 17 hours at 37℃，180 r/min.Then 0.70 g fresh mycelium，produced under the above conditions，was treated with a complex enzyme system comprising 1.50% lyase，0.50% snailase and 0.20% lysozyme for 3.25 h at 37℃.Under these conditions the regeneration rate of protoplasts was up to 36%.In addition，the laeA and cexA genes of Aspergillus niger TNA09 were successfully knocked out by protoplast transformation mediated by polyethyleneglycol（PEG）-CaCl2，laying a solid foundation for subsequent molecular transformations of industrial citric acid producing strains.