黑曲霉转录因子ACE1的功能分析及其对β-葡萄糖苷酶表达合成的影响

Functions of the transcription factor ACE1 in Aspergillus niger and its effect on the expression and synthesis of β-glucosidase

摘要:

【背景】 ACE1转录因子广泛存在于产纤维素酶的丝状真菌中，然而其在黑曲霉(Aspergillus niger)中的调控功能有待明确。【目的】 在A. niger中敲除和过表达转录因子ACE1，探究其对β-葡萄糖苷酶(β-glucosidase, BGL)表达、菌丝发育和色素合成的调控作用。【方法】 以A. niger An-1为出发菌株，利用CRISPR-Cas9基因编辑技术，获得敲除菌株Δace1、过表达菌株Oace1。于不同诱导环境下，检测各菌株中的BGL酶活变化、菌丝生长、产孢情况及黄色素合成差异。【结果】 系统发育树分析发现，A. niger与里氏木霉(Trichoderma reesei)、粗糙脉孢菌(Neurospora crassa)中ACE1的同源性较低。经原生质体转化法将操作质粒和供体片段同时导入，获得缺失突变菌株Δace1。以微晶纤维素、玉米芯粉为诱导物，纤维二糖为检测底物时，菌株Δace1的BGL酶活分别下降了86.1%、54.9%；以p-硝基苯酚-α-D-吡喃葡萄糖苷为检测底物时，菌株Δace1的BGL酶活分别下降了23.7%、63.98%。组装过表达框P_gpd-ace1-T_gla，在kusA位点进行精准插入，成功获得过表达菌株Oace1。以微晶纤维素诱导时，过表达菌株Oace1的BGL酶活是野生菌株An-1的1.37-2.16倍。在以甜菊糖苷和微晶纤维素为碳源的培养基上，缺失菌株Δace1的菌丝延伸迟缓、分生孢子梗分化延迟，而过表达菌株Oace1与菌株An-1的菌丝形态无差异。在以甜菊糖苷为碳源，进行固、液体培养时，敲除菌株Δace1生物合成黄色素的能力显著加强。【结论】 转录因子ACE1正向调控A. niger的BGL表达和产孢能力，负调控黄色素的合成，增强ACE1表达水平能够有效提升A. niger产BGL能力，本研究为优化A. niger的产酶性能提供新思路。

Abstract:

[Background] The transcription factor ACE1 is widely present in cellulase- producing filamentous fungi, while its regulatory function in Aspergillus niger remains unclear. [Objective] To investigate the regulatory effects of ACE1 on β-glucosidase (BGL) expression, mycelial development, and pigment biosynthesis by deleting and overexpressing ACE1 in A. niger. [Methods] The knockout strain Δace1 and overexpression strain Oace1 were constructed with the parent strain An-1 through CRISPR-Cas9 gene editing. The BGL activity, mycelial growth, sporulation, and yellow pigment were determined under various induction conditions. [Results] The phylogenetic analysis revealed low homology of ace1 between A. niger and Trichoderma reesei or Neurospora crassa. The mutant Δace1 was successfully constructed after introduction of the CRISPR-Cas9 plasmid and the donor fragment. When microcrystalline cellulose (MCC) and corncob powder were used as inducers, the BGL activity of Δace1 with cellobiose as the substrate decreased by 86.1% and 54.9%, and that of Δace1 with p-nitrophenyl-α-D-glucopyranoside (pNPG) as the substrate decreased by 23.7% and 63.98%, respectively. The overexpression cassette P_gpd-ace1-T_gla was precisely integrated into the kusA locus to construct Oace1. The BGL activity of Oace1 was 1.37-2.16 times that of the wild strain An-1 with MCC as the inducer. On the medium with stevioside or MCC as the carbon source, Δace1 showed delayed hyphal extension and conidiophore differentiation, while Oace1 retained normal morphology. The yellow pigment biosynthesis ability of Δace1 was significantly enhanced in the solid and liquid media containing stevioside. [Conclusion] The transcription factor ACE1 positively regulates BGL expression and sporulation, while it negatively regulates yellow pigment synthesis in A. niger. Strengthening the expression of ACE1 effectively improves BGL production. This study provides a new strategy for optimizing the enzyme production performance of A. niger.