优化黑曲霉柠檬酸生产菌株原生质体形成条件及表达系统的建立

Protoplasting Improvement of Industrial Citrate-Producing Aspergillus niger for DNA Transformation

优化了黑曲霉柠檬酸生产菌株的原生质体形成条件并建立基因表达系统。利用酶解法制备原生质体,最优的酶解液配比为5 mg/m L溶壁酶、0.2 U/m L几丁质酶和460 U/m L葡萄糖醛酸酶。最优的渗透压稳定剂为0.7 mol/L KCl,菌丝层厚度50μm,菌体量为0.003 g/m L。在培养基中添加1 mmol/L Mn2+有助于减少原生质体形成所需的酶量,在培养基中添加葡萄糖醛酸酶有助于得到独立的孢子进行萌发,从而促进原生质体的形成。利用共转化的方式,可以同时将两个表达框整合到基因组上并实现基因表达。

This article aimed to optimize protoplast formation conditions of industrial citrate-producing Aspergillus niger to establish genetic transformation system. As protoplasts were formed by enzyme digestion,components o f enzyme mixtures, digestion conditions and hyphae growth conditions were studied. The optimized enzyme mixture contained 5 mg/mL lysing enzyme, 0.2 U/mL chitinase and 460 U/mL glucuronidase. The best conditions for protoplasting were using 0.7 M KC1 as osmotic stabilizer, thickness of pellets hyphae for 50 μm ,cell weight o f 0.003 g/ mL. Furthermore, addition of Mn^2＋ in culture medium helped reduce the chitinase concentration necessary for protoplasting. In addition, glucuronidase in culture medium facilitated conidia separation during germination,further improved protoplast formation. Finally,two cassettes were inserted into genome simultaneously.