Effects of Transcription Factor CREA Knockout on the Morphology and the Secretion of β-glucosidase in Aspergillus niger

doi:10.13560/j.cnki.biotech.bull.1985.2024-1125

Abstract

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.

Key words: Aspergillus niger, β-glucosidase, transcription factor, CRISPR-Cas9, gene knockout, carbon catabolite repression

CHENG Hui-juan, WANG Xin, SHI Xiao-tao, MA Dong-xu, GONG Da-chun, HU Jun-peng, XIE Zhi-wen. Effects of Transcription Factor CREA Knockout on the Morphology and the Secretion of β-glucosidase in Aspergillus niger[J]. Biotechnology Bulletin, 2025, 41(6): 344-354.

Figures/Tables 9

Table 1 Primers used in this study

Name	Primer sequence（5′-3′）
5srRNA-F	TACTGGGCCCGGGAAGATCTGGTTGGAGATTCCAGACTCAG
5srRNA-R	CCTTGTCTGCTTACGCGATCACATACAACAGAAGGGATTC
sgRNA-F	GATCGCGTAAGCAGACAAGGGTTTTAGAGCTAGAAATAGC
sgRNA-R	CATGGTCATAGCTGTTTCCGAAAAAAGCACCGACTCGG
pFC332-F	ACCCTGTGAGTCTTGATAGAC
pFC332-R	CCGTTCGAGAGCATGATC
gtsy-F	ACTTATTACCCCGGGGTCCA
gtsy-R	CTGGACGGAGTAGAAGGGG
gtxy-F	TACACATGAATTTGATATAAAGGTCTGATGCC
gtxy-R	ACCCCTTCTACTCCGTCCAGGCGAATTCCGTTGCCGG
creAYZ-F	CAACATCACCCTTGCCCGAC
18S-F	ACTCACCAGGTCCAGACAAAATAAG
18S-R	AAGCAGACAAATCACTCCA
bglA-F	CAGGATGCCTCAGACTATCTTC
bglA-R	CGCCAAGAGAAACATACAGTTG
cbhA-F	CAGTTGTCATTGACTCGAACTG
cbhA-R	TTTCAGTGTCAATGAATCACCG
eglA-F	ACGATGTGCTCTCAATATGACA
eglA-R	ATCACTCACGAGCTTCTTGTTA

Fig. 1 Evolutionary tree analysis of CREA amino acid sequencesAoCreA: CREA in Aspergillus oryzae; AnCreA:CREA in Aspergillus niger; AfCreA: CREA in Aspergillus fumigatus; AaCreA: CREA in Aspergillus aculeatus; TrCreA: CREA in Trichoderma reesei; TgCreA: CREA in Trichoderma gamsii; AdCreA: CREA in Aspergillus nidulans; PdCreA: CREA in Penicillium decumens; PoCreA: CREA in Penicillium oxalicum; NcCreA: CREA in Neurospora crassa

Fig. 2 Construction of CRISPR-Cas9 targeting plasmid and donor DNAA: Construction of sgRNA expression cassette; B: construction of pFC332-target-sgRNA; C: construction of DNA donor; D-F: PCR detection of sgRNA expression cassette, pFC332-target-sgRNA and DNA donor, respectively

Fig. 3 Knockout of the creA gene in A. nigerA: Schematic diagram of creA gene editing and verification of PCR knockout; B:Sequencing imageof creA gene knockout

Fig. 4 Differences in the morphologies of A. niger in different mediaA: Cell morphology in PDA solid plates cultured for 48/60/72 h, respectively. B: Cell morphology in esculin solid plates cultured for 48/60/72 h, respectively. C: Cell morphology in liquid growth medium

Fig. 5 Effects of creA knockout on the enzymatic activities of different enzymesA: BGL enzyme activity determined by cellobiose as substrate. B: BGL enzyme activity assayed with p-NPG as substrate. C: CBH enzyme activity assayed with p-NPC as substrate. D: EG enzyme activity assayed by CMC-Na as substrate

Fig. 6 Effects of creA knockout on expression and proteinA: Transcription levels of bglA, cbh1, and egl1 genes; B: SDS-PAGE on the 7 th day of fermentation, 1-3 refer to the fermented samples of △creA strain, and 4-6 refer to the fermentation samples of wild strains. C: Protein content and specific activity of BGL (cellobiose as substrate) on the fermentation of day 7

Fig. 7 Effects of creA knockout on BGL production by A. niger at different glucose concentrationsA: Enzyme activity level after 4 d of fermentation. B: Enzyme activity level after 5 d of fermentation. C: Enzyme activity level after 6 d of fermentation. D: Enzyme activity level for 7 d of fermentation

Fig. 8 Knockout of creA promotes the production of BGL in A. niger

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