Nie Hongmei, Wang Zebin, Lin Zhenkai, Gao Yan, Zhang Yinjun, Zheng Jianyong, Cheng Yong
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, People's Republic of China.
Biotechnol Lett. 2025 Sep 25;47(5):114. doi: 10.1007/s10529-025-03657-6.
The engineered Aspergillus niger strain AnCALB005 was selected as the research strain, which is a high-yield strain of Candida antarctica B lipase constructed in our laboratory. CRISPR/Cas9-mediated gene knockout was employed to construct the multiple protease-deficient strains targeting five genes (pepA, pepB, pepC, pepE and pepF) in the A. niger AnCALB005. Among the engineered variants, a triple-knockout strain lacking pepA, pepB, and pepF demonstrated 56% enhanced hydrolytic lipase activity relative to the parental strain. Fermentation culture conditions were initially screened through single-factor experiments. Building on these results, critical parameters were statistically determined via Plackett-Burman (PB) design. This was followed by a steepest ascent method combined with Box-Behnken (BB) response surface methodology. Key factors influencing lipase production (identified as maltose concentration, corn steep concentration, and shaking speed) were optimized. The final optimized fermentation conditions comprised: maltose (52 g/L), corn steep (52 g/L), KHPO (5 g/L), soybean cake flour (30 g/L), initial pH 6.5, inoculation amount 10% (v/v), and shaking speed 220 rpm. Under the optimized fermentation conditions, Shake-flask validation of the engineered A. niger yielded a lipase activity of 46.66 U/mL, representing an increase of 92.01%. Scale-up fermentation in a 5 L bioreactor applying these optimized conditions over 120 h of cultivation achieved a lipase activity of 79.31 U/mL.
工程黑曲霉菌株AnCALB005被选为研究菌株，它是我们实验室构建的南极假丝酵母B脂肪酶高产菌株。采用CRISPR/Cas9介导的基因敲除技术构建了针对黑曲霉AnCALB005中五个基因（pepA、pepB、pepC、pepE和pepF）的多蛋白酶缺陷菌株。在工程变体中，缺失pepA、pepB和pepF的三基因敲除菌株相对于亲本菌株表现出56%的水解脂肪酶活性增强。最初通过单因素实验筛选发酵培养条件。基于这些结果，通过Plackett-Burman（PB）设计统计确定关键参数。随后采用最速上升法结合Box-Behnken（BB）响应面法。对影响脂肪酶产生的关键因素（确定为麦芽糖浓度、玉米浆浓度和摇床转速）进行了优化。最终优化的发酵条件包括：麦芽糖（52 g/L）、玉米浆（52 g/L）、KHPO（5 g/L）、豆饼粉（30 g/L）、初始pH 6.5、接种量10%（v/v）和摇床转速220 rpm。在优化的发酵条件下，对工程化黑曲霉进行摇瓶验证，得到的脂肪酶活性为46.66 U/mL，增幅为92.01%。在5 L生物反应器中应用这些优化条件进行120小时的扩大培养发酵，脂肪酶活性达到79.31 U/mL。
