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<p>The engineered <i>Aspergillus niger</i> strain <i>An</i>CALB005 was selected as the research strain, which is a high-yield strain of <i>Candida antarctica</i> B lipase constructed in our laboratory. CRISPR/Cas9-mediated gene knockout was employed to construct the multiple protease-deficient strains targeting five genes (<i>pepA</i>, <i>pepB</i>, <i>pepC</i>, <i>pepE</i> and <i>pepF</i>) in the <i>A. niger An</i>CALB005. Among the engineered variants, a triple-knockout strain lacking <i>pepA</i>, <i>pepB</i>, and <i>pepF</i> 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), K<sub>2</sub>HPO<sub>4</sub> (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 <i>A. niger</i> 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&#xa0;h of cultivation achieved a lipase activity of 79.31 U/mL.</p>

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Enhancing lipase activity in Aspergillus niger through CRISPR/Cas9-mediated protease gene knockout and fermentation optimization

  • 期刊论文

  • 发表日期：

    2025年9月25日

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  • Hongmei Nie,
  • Zebin Wang,
  • Zhenkai Lin,
  • Yan Gao,
  • Yinjun Zhang,
  • Jianyong Zheng,
  • Yong Cheng

摘要

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), K2HPO4 (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.