生物技术通报2025，Vol.41Issue(6)：344-354,11.DOI:10.13560/j.cnki.biotech.bull.1985.2024-1125
转录因子CREA敲除对黑曲霉形态和分泌β-葡萄糖苷酶的影响
Effects of Transcription Factor CREA Knockout on the Morphology and the Secretion of β-glucosidase in Aspergillus niger
摘要
Abstract
[Objective]Transcription factor CREA in Aspergillus niger was knocked out to explore its regulatory effects on hyphal development and BGL expression,which may provide effective regulatory targets for the design of high-yield BGL cell factories.[Method]The A.niger An-1 was used as the research strain,and the operation plasmid and donor fragment for the knockout creA gene were constructed.After protoplast transformation,resistance screening,and mycelial PCR verification,the knockout strain ΔcreA was obtained using the CRISPR-Cas9 genome editing technology.The effect of the transcription factor CREA on the fungus morphology and fermentation performance was examined by plate culture and shaking flask fermentation,respectively.The impact of carbon catabolite repression on BGL biosynthesis was explored under different concentrations of glucose.[Result]The creA gene in the A.niger An-1 was precisely knocked out,and the deletion mutant strain ΔcreA was obtained.Compared with the wild-type strain,the colony morphology of the creA strain showed a wheel-like wrinkle on potato dextrose agar and stronger β-glucosidase solid secretion ability on the esculin color plate.With cellobiose and p-nitrophenyl-α-D-glucopyranoside as substrates,the BGL activity of the ΔcreA strain was 1.5 times and 1.8 times higher than that of the wild-type strain,respectively.Fluorescence quantitative PCR showed that the knockout of creA increased the expression of the bglA gene by 8.4 times.During the fermentation process with different concentrations of glucose added,the enzyme activity of the ΔcreA strain was higher than that of the wild-type,showing the derepression of carbon catabolite repression.[Conclusion]The transcription factor CREA regulates the hyphal growth and BGL expression of A.niger An-1.Its knockout alleviates the carbon catabolite repression and enhances the ability of A.niger-An to produce BGL.This study provides theoretical support for regulatory modification of A.niger to optimize the production performance of BGL.关键词
黑曲霉/β-葡萄糖苷酶/转录因子/CRISPR-Cas9/基因敲除/碳阻遏效应Key words
Aspergillus niger/β-glucosidase/transcription factor/CRISPR-Cas9/gene knockout/carbon catabolite repression引用本文复制引用
程慧娟,王昕,石小涛,马东旭,龚大春,胡骏鹏,谢智文..转录因子CREA敲除对黑曲霉形态和分泌β-葡萄糖苷酶的影响[J].生物技术通报,2025,41(6):344-354,11.基金项目
湖北省教育厅科学技术研究计划青年人才项目(Q20231201),湖北省自然科学基金创新发展联合基金项目(2024AFD178) （Q20231201）
