柠檬酸工业生产菌黑曲霉TNA09基因敲除体系的构建

Construction of A Gene Knockout System in An Industrial Citric Acid Producing Strain of Aspergillus niger TNA09

黑曲霉TNA09是高产柠檬酸的工业生产菌株,由于经过多次物理和化学诱变筛选,所以对其进行传统遗传改造非常困难。该试验成功构建黑曲霉TNA09原生质体转化方法,实现该工业菌株的基因工程操作。通过试验得出原生质体制备和再生的最佳条件:取CM斜面培养5 d的新鲜孢子约1×10^(8)个至100 mL马铃薯葡萄糖肉汤(potato dextrose broth,PDB)液体培养基,于37℃、180 r/min摇床培养17 h,取幼嫩菌丝0.70 g至1.50%裂解酶+0.50%蜗牛酶+0.20%溶菌酶的复合酶体系中,37℃处理3.25 h,该条件下原生质体再生率可达36%,并利用聚乙二醇(polyethyleneglycol,PEG)-CaCl_(2)介导的原生质体转化方法一次成功将黑曲霉laeA、cexA基因敲除,为后续的柠檬酸工业菌株的分子改造奠定了坚实基础。

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 1×10^(8) Aspergillus 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)-CaCl_(2),laying a solid foundation for subsequent molecular transformations of industrial citric acid producing strains.