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

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

摘要/Abstract

摘要：

目的在黑曲霉中敲除转录因子CREA，探究其对菌丝发育和β-葡萄糖苷酶（β-glucosidase, BGL）表达的调控作用，为开发高产BGL的细胞工厂提供有效的调控改造靶点。方法以黑曲霉菌株An-1为研究对象，构建敲除creA基因的操作质粒和供体片段。经原生质体转化、抗性筛选和菌丝PCR验证，利用CRISPR-Cas9基因编辑技术，获得敲除菌株ΔcreA。通过平板培养和摇瓶发酵，分别考察转录因子CREA对菌体形态和产BGL的影响，并在不同浓度葡萄糖下，探究碳阻遏效应对BGL生物合成的影响。结果在黑曲霉菌株An-1中对creA基因进行了精准敲除，获得缺失突变菌株ΔcreA。与野生型菌株相比，ΔcreA菌株的菌落形态呈现车轮状褶皱，在七叶苷显色平板上表现出较强的β-葡萄糖苷酶分泌能力。以纤维二糖和p-硝基苯酚-α-D-吡喃葡萄糖苷为底物，ΔcreA菌株的BGL活性分别为野生型菌株的1.5倍和1.8倍。荧光定量PCR显示，creA的敲除使bglA基因的表达量提升了8.4倍。在添加不同浓度葡萄糖的发酵过程中，ΔcreA菌株的酶活水平始终高于野生型，表现出脱碳阻遏效应。结论 CREA转录因子调控黑曲霉的菌丝发育和BGL表达，其敲除减轻了碳阻遏效应，提高了黑曲霉产BGL的能力，为调控改造黑曲霉以优化BGL的生产性能提供了理论支持。

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

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

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.

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.

图/表 9

表1 本研究所用引物

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

图1 CREA氨基酸序列进化树分析AoCreA：米曲霉的CREA； AnCreA：黑曲霉的CREA； AfCreA：烟曲霉的CREA； AaCreA：棘孢曲霉的CREA；TrCreA：里氏木霉的CREA；TgCreA：盖姆斯木霉的CREA；AdCreA：构巢曲霉的CREA；PdCreA：斜卧青霉的CREA；PoCreA：草酸青霉的CREA；NcCreA：粗糙脉孢菌的CREA

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

图2 CRISPR-Cas9靶向质粒及供体DNA的构建A：sgRNA表达盒的构建；B：pFC332-target-sgRNA的构建；C：无选择标记供体DNA片段的构建；D-F：sgRNA表达盒、pFC332-target-sgRNA质粒和供体DNA片段的PCR检测

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

图3 黑曲霉中creA基因的敲除A：creA基因编辑示意图及PCR敲除验证；B：creA基因敲除测序图

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

图4 黑曲霉菌体在不同培养基中形态的差异A：PDA固体平板分别培养48/60/72 h的菌体形态；B：七叶苷固体平板分别培养48/60/72 h的菌体形态；C：液体生长培养基菌体形态

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

图5 creA的敲除对不同酶酶活的影响A：纤维二糖为底物测定BGL酶活；B：p-NPG为底物测定BGL酶活；C：p-NPC为底物测定CBH酶活；D：CMC-Na为底物测定EG酶活

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

图6 creA的敲除对表达量及蛋白的影响A：bglA、cbh1、egl1基因的转录水平；B：第7天发酵液上清的SDS-PAGE，1-3代表△creA菌株发酵样品，4-6代表野生菌株发酵样品；C：第7天发酵液的上清蛋白含量及BGL比酶活（以纤维二糖为底物）

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

图7 不同葡萄糖浓度下creA的敲除对黑曲霉产BGL的影响A：发酵4 d的酶活水平；B：发酵5 d的酶活水平；C：发酵6 d的酶活水平；D：发酵7 d的酶活水平

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

图8 敲除creA促进黑曲霉发酵生产BGL

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

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