Biotechnol Lett. 2025 Sep 25;47(5):114. doi: 10.1007/s10529-025-03657-6.
ABSTRACT
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.
PMID:40996539 | DOI:10.1007/s10529-025-03657-6
