β-葡萄糖苷酶(β-glucosidase,BGL)作为重要的糖苷水解酶,不仅可用于功能性活性物质的生产,改善食品风味、提升食品营养价值,还是新一代工业发酵用碳源秸秆糖生产的关键限速酶,具有重要的应用价值。为了有效改造自主筛选的非模式黑曲霉An-1产BGL活力,该研究系统优化了黑曲霉An-1原生质体的制备条件与再生方法,建立了稳定的黑曲霉An-1遗传转化体系,具体为:将固体平板培养的菌丝作为酶解材料,使用混合酶(1%纤维素+0.5%蜗牛酶+0.25%溶菌酶,质量分数)处理2 h;在STC缓冲液孵育后,采用直接抗性筛选的方式,转化子的阳性率高达100%;通过比较不同的破壁方式对菌丝PCR的影响,确定了碱裂解法的破菌效果最佳,以其裂解液作为模版的检测效率最高达97%。然后以红色荧光蛋白作为报告基因,以Pgpd为启动子和Tgla为终止子,成功过表达了带有报告基因的11条BGL基因,筛选出黑曲霉An-1的关键BGL为HQM9510。以pFC332-Cas9-sgRNA为质粒,以黑曲霉An-1基因组为模板,以kus-up-F和kus-down-R为引物获取供体片段,精准敲除kusA基因,建立了非模式黑曲霉An-1的基因组编辑体系。所构建的过表达HQM9510基因的工程菌株O9510,以纤维二糖和p-硝基苯酚-α-D-吡喃葡萄糖苷为底物的摇瓶酶活力分别提升至12.88 U/mL和17.10 U/mL,实现了BGL的高效表达。该研究为分子改造黑曲霉An-1以提升其产酶能力奠定了科学基础。
β-Glucosidase (BGL), as an important glycoside hydrolase, plays a crucial role in synthesizing functional active substances, improving food flavor, and enhancing the nutritional value of food.BGL is also a key rate-limiting enzyme for the preparation of sugar from straw to provide a next-generation carbon source for industrial fermentation.To effectively enhance the BGL activity, this study systematically optimized the preparation conditions and regeneration methods for the protoplasts and established a stable genetic transformation system for Aspergillus niger An-1.The optimal conditions of protoplast preparation were to use the mycelium cultured on solid plates as the enzymatic material and treat it with a mixture of enzymes (1% cellulose+0.5% snail enzyme+0.25% lysozyme) for 2 hours.After incubating the transformation system in STC buffer, the positive rate of transformants during resistance screening reached 100%.Subsequently, different cell wall-breaking methods were compared to optimize the efficiency of mycelial PCR.The alkaline lysis method had the highest detection efficiency of 97% when its lysate was used as the template.HQM9510 was identified as the key BGL of A.niger An-1 via the overexpression of eleven BGL genes with the red fluorescent protein as the reporter gene, Pgpd as the promoter and Tgla as the terminator.The kusA gene on the genome was precisely knocked out when the plasmid pFC332-Cas9-sgRNA and linear knockout donor (kus-up-F and kus-down-R as primers) were co-transformed into A.niger An-1. The O9510 strain overexpressing HQM9510 was obtained.With cellobiose and p-nitrophenyl-α-D-glucopyranoside as substrates, the BGL activity of O9510 reached 12.88 U/mL and 17.10 U/mL.This study lays a scientific foundation for the molecular engineering of A.niger An-1 to enhance its enzyme production capacity.
[1] PARK M, JEONG M K, KIM M, et al.Modification of isoflavone profiles in a fermented soy food with almond powder[J].Journal of Food Science, 2012, 77(1):C128-C134.
[2] KANNAN P, SHAFREEN M M, ACHUDHAN A B, et al.A review on applications of β-glucosidase in food, brewery, pharmaceutical and cosmetic industries[J].Carbohydrate Research, 2023,530:108855.
[3] FITZPATRICK J, KRICKA W, JAMES T C, et al. Expression of three Trichoderma reesei cellulase genes in Saccharomyces pastorianus for the development of a two-step process of hydrolysis and fermentation of cellulose. Journal of Applied Microbiology,2014 ,117(1):96-108.
[4] ZHENG X M, CAIRNS T, ZHENG P, et al.Protocol for gene characterization in Aspergillus niger using 5S rRNA-CRISPR-Cas9-mediated Tet-on inducible promoter exchange[J].STAR Protocols, 2022, 3(4):101838.
[5] LIU D D, LIU Q, GUO W Z, et al.Development of genetic tools in glucoamylase-hyperproducing industrial Aspergillus niger strains[J].Biology, 2022,11(10):1396.
[6] LI Y Y, LI C, MUHAMMAD AQEEL S, et al.Enhanced expression of xylanase in Aspergillus niger enabling a two-step enzymatic pathway for extracting β-glucan from oat bran[J].Bioresource Technology, 2023, 377:128962.
[7] DONG L B, YU D, LIN X T, et al.Improving expression of thermostable trehalase from Myceliophthora sepedonium in Aspergillus niger mediated by the CRISPR/Cas9 tool and its purification, characterization[J].Protein Expression and Purification, 2020, 165:105482.
[8] CAI L N, XU S N, LU T, et al.Directed expression of halophilic and acidophilic β-glucosidases by introducing homologous constitutive expression cassettes in marine Aspergillus niger[J].Journal of Biotechnology, 2019, 292:12-22.
[9] WANG L, XIE Y J, CHANG J J, et al.A novel sucrose-inducible expression system and its application for production of biomass-degrading enzymes in Aspergillus niger[J].Biotechnology for Biofuels.2023, 16(23):1-20.
[10] 龚大春, 马东旭, 王昕, 等.一种高产BGL黑曲霉菌及其构建方法与应用:中国, CN 202510036900X[P].2025-01-09.
GONG D C, MA D X, WANG X, et al.A high-yield BGL Aspergillus niger strain, its construction method and application:China, CN 202510036900X[P].2025-01-09.
[11] 赵君. 黑曲霉胞β-葡萄糖苷酶的鉴定及其在纤维素降解酶系合成调控中的作用[D].郑州:河南农业大学,2021.
ZHAO J.Identification of intracellular β-glucosidase from Aspergillus niger and its role in the synthesis and regulation of cellulolytic enzyme system[D].Zhengzhou:Henan Agricultural University, 2021.
[12] LI Z H, YAO G S, WU R M, et al.Synergistic and dose-controlled regulation of cellulase gene expression in Penicillium oxalicum[J].PLoS Genetics, 2015, 11(9):e1005509.
[13] QIAN Y C, ZHONG L X, GAO J, et al.Production of highly efficient cellulase mixtures by genetically exploiting the potentials of Trichoderma reesei endogenous cellulases for hydrolysis of corncob residues[J].Microbial Cell Factories, 2017, 16(1):207-223.
[14] 朱思远.无抗黑曲霉表达系统构建及脂肪酶的表达[D].无锡:江南大学, 2019.
ZHU S Y.Construction of non-resistant Aspergillus niger expression system and expression of lipase[D].Wuxi:Jiangnan University, 2019.
[15] 应汉杰,张涛,陈勇, 等.一株重组黑曲霉基因工程菌及构建方法和应用:中国,CN202110095568.6[P].2020-07-31.
YING H J, ZHANG T, CHEN Y, et al.A recombinant Aspergillus niger genetically engineered strain, construction method and application:China, CN202110095568.6[P].2020-07-31.
[16] 张晓娟,刘松青,夏珊, 等.一种用于PCR扩增的丝状真菌DNA快速提取方法:中国, CN201510918551.0[P].2016-02-10.
ZHANG X J, LIU S Q, XIA S, et al.A rapid extraction method for filamentous fungal DNA for PCR amplification:China, CN201510918551.0[P].2016-02-10.
[17] 赵杰宏, 桂艳玲, 韩洁, 等.一种丝状真菌快速PCR模板的简易制备方法:中国,CN202110031221.5[P].2021-05-18.
ZHAO J H, GUI Y L, HAN J, et al.A simple method for preparing a rapid PCR template for filamentous fungi:China, CN202110031221.5[P].2021-05-18.
[18] 罗中钦, 程琳, 张茜, 等.丝状真菌PCR模板DNA的快速制备方法[J].生物技术通报, 2015(9):79-83.
LUO Z Q, CHENG L, ZHANG X, et al.A rapid method of preparing DNA template of filamentous fungi for PCR amplification[J].Biotechnology Bulletin, 2015(9):79-83.
[19] KLUGE J, TERFEHR D, KÜCK U.Inducible promoters and functional genomic approaches for the genetic engineering of filamentous fungi[J].Applied Microbiology and Biotechnology, 2018, 102(15):6357-6372.
[20] ZHENG X M, ZHENG P, ZHANG K, et al.5S rRNA Promoter for guide RNA expression enabled highly efficient CRISPR/Cas9 genome editing in Aspergillus niger[J].ACS Synthetic Biology, 2019, 8(7):1568-1574.
[21] DONG L B, LIN X T, YU D, et al.High-level expression of highly active and thermostable trehalase from Myceliophthora thermophila in Aspergillus niger by using the CRISPR/Cas9 tool and its application in ethanol fermentation[J].Journal of Industrial Microbiology & Biotechnology, 2020, 47(1):133-144.
[22] ZHAO J, SHI D C, YANG S, et al.Identification of an intracellular β-glucosidase in Aspergillus niger with transglycosylation activity[J].Applied Microbiology Biotechnology, 2020, 104(19):8367-8380.
[23] PANG A P, WANG H Y, LUO Y S, et al.Investigating the cellular functions of β-glucosidases for synthesis of lignocellulose-degrading enzymes in Trichoderma reesei[J].Engineering Microbiology, 2023, 3(4):100105.
[24] GAO J, QIAN Y C, WANG Y F, et al.Production of the versatile cellulase for cellulose bioconversion and cellulase inducer synthesis by genetic improvement of Trichoderma reesei[J].Biotechnology for Biofuels, 2017, 10:272.
[25] 阚钦标.草酸青霉β-葡萄糖苷酶的过表达及纤维素酶新调控位点的筛选[D].济南:山东大学,2016.
KAN Q B.Penicillium oxalate Kan QB.Overexpression of β-glucosidase of Penicillium oxalate and screening of new regulatory sites for cellulase [D].Jinan:Shandong University, 2016.
