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CRISPR/Cas9-mediated Inactivation of the Pectinase Gene in Aspergillus niger and Evaluation of the Mutant Strain |
WANG Yan-mei,KOU Hang,MA Mei,SHEN Yu-yu,ZHAO Bao-ding,LU Fu-ping,LI Ming() |
Key Laboratory of Industrial Fermentation Microbiology (Tianjin University of Science and Technology), Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China |
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Abstract Pectinase preparations have been widely used in China. However, efficient and specific pectinases are still lacking in the market. Producing a single-component pectinase using modified Aspergillus niger by genetic engineering technology has become an effective solution to meet the growing industrial demand of pectinase. Constructing an efficient CRISPR-Cas9 technology can provide an efficient genome editing tool for the construction of Aspergillus niger chassis strain with high yield single pectinase. First, the pyrG gene on the genome of pectinase-producing Aspergillus niger was knocked out to construct the uracil auxotrophic strain AnΔpyrG, and the Cas9 expression cassette and pyrG expression cassette were integrated precisely at the pyrG site of the AnΔpyrG strain to construct AnCas9 strain constitutively expressing the Cas9 gene. Then, the pLM2-sgRNA plasmid which contained the gpdA promoter, hammerhead ribozyme, and HDV ribozymes for the efficient expression and maturation of sgRNA was constructed. And finally, the CRISPR-Cas9 gene editing system is successfully established. The 4978020 and the 4983861 gene were used to detect the gene editing efficiency of the constructed CRISPR-Cas9 system, and the phenotypic changes and enzyme production changes of the 4978020 gene function deletion strain were detected. The results showed that the pectinase gene editing efficiency is more than 50%, the phenotype and pectinase activity of the strain AnΔ4978020 had no significant changes compared with the original strain. An efficiency CRISPR-Cas9 system was successfully constructed in Aspergillus niger producing pectinase, and losing of the 4978020 gene function did not affect the phenotype of the strain, which laid the foundation for the construction of high yield single pectinase Aspergillus niger chassis strain.
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Received: 01 March 2021
Published: 01 June 2021
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Corresponding Authors:
Ming LI
E-mail: liming09@tust.edu.cn
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[1] |
Mohnen D. Pectin structure and biosynthesis. Current Opinion in Plant Biology, 2008,11(3):266-277.
doi: 10.1016/j.pbi.2008.03.006
|
|
|
[2] |
Lara-Márquez A, Zavala-Páramo M G, López-Romer E, et al. Biotechnological potential of pectinolytic complexes of fungi. Biotechnology Letters, 2011,33(5):859-868.
doi: 10.1007/s10529-011-0520-0
pmid: 21246254
|
|
|
[3] |
Willats W G T, McCartney L, Mackie W, et al. Pectin: cell biology and prospects for functional analysis. Plant Molecular Biology, 2001,47(1-2):9-27.
doi: 10.1023/A:1010662911148
|
|
|
[4] |
傅海, 赵佳, 李伟, 等. 果胶酶研究进展及应用. 生物化工, 2020,6(5):148-150,153.
|
|
|
[4] |
Fu H, Zhao J, Li W, et al. Research progress and application of pectinase. Biological Chemical Engineering, 2020,6(5):148-150,153.
|
|
|
[5] |
Alkorta I, Garbisu C, Llama M J, et al. Industrial applications of pectic enzymes: a review. Process Biochemistry, 1998,33(1):21-28.
doi: 10.1016/S0032-9592(97)00046-0
|
|
|
[6] |
Mieszczakowska-Frᶏc M, Markowski J, Zbrzeźniak J, et al. Impact of enzyme on quality of blackcurrant and plum juices. LWT - Food Science and Technology, 2012,49(2):251-256.
doi: 10.1016/j.lwt.2011.12.034
|
|
|
[7] |
Wang W D, Xu S Y, Jin M K. Effects of different maceration enzymes on yield, clarity and anthocyanin and other polyphenol contents in blackberry juice. International Journal of Food Science & Technology, 2009,44(12):2342-2349.
|
|
|
[8] |
Kohli P, Gupta R. Alkaline pectinases: a review. Biocatalysis and Agricultural Biotechnology, 2015,4(3):279-285.
doi: 10.1016/j.bcab.2015.07.001
|
|
|
[9] |
Saoudi B, Habbeche A, Kerouaz B, et al. Purification and characterization of a new thermoalkaliphilic pectate lyase from Actinomadura keratinilytica Cpt20. Process Biochemistry, 2015,50(12):2259-2266.
doi: 10.1016/j.procbio.2015.10.006
|
|
|
[10] |
Hoondal G, Tiwari R, Tewari R, et al. Microbial alkaline pectinases and their industrial applications: a review. Applied Microbiology and Biotechnology, 2002,59(4-5):409-418.
doi: 10.1007/s00253-002-1061-1
|
|
|
[11] |
Wang X W, Lu Z H, Xu T, et al. Improving the specific activity and thermo-stability of alkaline pectate lyase from Bacillus subtilis 168 for bioscouring. Biochemical Engineering Journal, 2018,129:74-83.
doi: 10.1016/j.bej.2017.11.001
|
|
|
[12] |
Barman S, Sit N, Badwaik L S, et al. Pectinase production by Aspergillus niger using banana (Musa balbisiana) peel as substrate and its effect on clarification of banana juice. Journal of Food Science and Technology, 2015,52(6):3579-3589.
|
|
|
[13] |
Mrudula S, Anitharaj R. Pectinase production in solid state fermentation by Aspergillus niger using orange peel as substrate. 2011
|
|
|
[14] |
Ann Ran F, Hsu P D, Wright J, et al. Genome engineering using the CRISPR-Cas9 system. Nature Protocols, 2013,8(11):2281-2308.
doi: 10.1038/nprot.2013.143
pmid: 24157548
|
|
|
[15] |
Schuster M, Kahmann R. CRISPR-Cas9 genome editing approaches in filamentous fungi and oomycetes. Fungal Genetics and Biology, 2019,130:43-53.
doi: 10.1016/j.fgb.2019.04.016
|
|
|
[16] |
Nødvig C S, Nielsen J B, Kogle M E, et al. A CRISPR-Cas9 system for genetic engineering of filamentous fungi. PLoS One, 2015,10(7):e0133085.
doi: 10.1371/journal.pone.0133085
|
|
|
[17] |
Pohl C, Kiel J A K W, Driessen A J M, et al. CRISPR/Cas9 based genome editing of Penicillium chrysogenum. ACS Synthetic Biology, 2016,5(7):754-764.
doi: 10.1021/acssynbio.6b00082
pmid: 27072635
|
|
|
[18] |
Platt R J. CRISPR tool modifies genes precisely by copying RNA into the genome. Nature, 2019,576(7785):48-49.
doi: 10.1038/d41586-019-03392-9
|
|
|
[19] |
Shi T Q, Gao J, Wang W J, et al. CRISPR/Cas9-based genome editing in the filamentous fungus Fusarium fujikuroi and its application in strain engineering for gibberellic acid production. ACS Synthetic Biology, 2019,8(2):445-454.
doi: 10.1021/acssynbio.8b00478
|
|
|
[20] |
Yang Y J, Singh R P, Lan X, et al. Genome editing in model strain Myxococcus xanthus DK1622 by a site-specific cre/loxP recombination system. Biomolecules, 2018,8(4):E137.
|
|
|
[21] |
李红花, 刘钢. CRISPR/Cas9在丝状真菌基因组编辑中的应用. 遗传, 2017,39(5):355-367.
|
|
|
[21] |
Li H H, Liu G. The application of CRISPR/Cas9 in genome editing of filamentous fungi. Hereditas, 2017,39(5):355-367.
|
|
|
[22] |
Michielse C B, Hooykaas P J J, van den Hondel C A M J J, et al. Agrobacterium -mediated transformation of the filamentous fungus Aspergillus awamori. Nature Protocols, 2008,3(10):1671-1678.
doi: 10.1038/nprot.2008.154
pmid: 18833205
|
|
|
[23] |
Sandri I G, Fontana R C, Moura da Silveira M. Influence of pH and temperature on the production of polygalacturonases by Aspergillus fumigatus. LWT - Food Science and Technology, 2015,61(2):430-436.
doi: 10.1016/j.lwt.2014.12.004
|
|
|
[24] |
Michielse C B, Hooykaas P J J, Hondel C A M J J, et al. Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Current Genetics, 2005,48(1):1-17.
doi: 10.1007/s00294-005-0578-0
|
|
|
[25] |
Weyda I, Yang L, Vang J, et al. A comparison of Agrobacterium-mediated transformation and protoplast-mediated transformation with CRISPR-Cas9 and bipartite gene targeting substrates, as effective gene targeting tools for Aspergillus carbonarius. Journal of Microbiological Methods, 2017,135:26-34.
doi: 10.1016/j.mimet.2017.01.015
|
|
|
[26] |
Kamijo J, Sakai K, Suzuki H, et al. Identification and characterization of a thermostable pectate lyase from Aspergillus luchuensis var. saitoi. Food Chemistry, 2019,276:503-510.
doi: S0308-8146(18)31824-7
pmid: 30409626
|
|
|
[27] |
Wang Q, Coleman J J. Progress and challenges: development and implementation of CRISPR/Cas9 technology in filamentous fungi. Computational and Structural Biotechnology Journal, 2019,17:761-769.
doi: 10.1016/j.csbj.2019.06.007
|
|
|
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