Browse > Article
http://dx.doi.org/10.1080/12298093.2020.1838115

Genetic Manipulation and Transformation Methods for Aspergillus spp.  

Son, Ye-Eun (School of Food Science and Biotechnology, Kyungpook National University)
Park, Hee-Soo (School of Food Science and Biotechnology, Kyungpook National University)
Publication Information
Mycobiology / v.49, no.2, 2021 , pp. 95-104 More about this Journal
Abstract
Species of the genus Aspergillus have a variety of effects on humans and have been considered industrial cell factories due to their prominent ability for manufacturing several products such as heterologous proteins, secondary metabolites, and organic acids. Scientists are trying to improve fungal strains and re-design metabolic processes through advanced genetic manipulation techniques and gene delivery systems to enhance their industrial efficiency and utility. In this review, we describe the current status of the genetic manipulation techniques and transformation methods for species of the genus Aspergillus. The host strains, selective markers, and experimental materials required for the genetic manipulation and fungal transformation are described in detail. Furthermore, the advantages and disadvantages of these techniques are described.
Keywords
CRISPR/Cas; protoplast; Agrobacterium; Aspergillus; homologous recombination;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Nielsen ML, de Jongh WA, Meijer SL, et al. Transient marker system for iterative gene targeting of a prototrophic fungus. Appl Environ Microbiol. 2007;73:7240-7245.   DOI
2 Nielsen JB, Nielsen ML, Mortensen UH. Transient disruption of non-homologous endjoining facilitates targeted genome manipulations in the filamentous fungus Aspergillus nidulans. Fungal Genet Biol. 2008;45:165-170.   DOI
3 Goswami RS. Targeted gene replacement in fungi using a split-marker approach. Methods Mol Biol. 2012;835:255-269.   DOI
4 Hutchison HT, Hartwell LH. Macromolecule synthesis in yeast spheroplasts. J Bacteriol. 1967;94:1697-1705.   DOI
5 Anne J, Eyssen H, Somer PD. Formation and regeneration of Penicillium chrysogenum protoplasts. Arch Microbiol. 1974;98:159-166.   DOI
6 Tilburn J, Scazzocchio C, Taylor GG, et al. Transformation by integration in Aspergillus nidulans. Gene. 1983;26:205-221.   DOI
7 Szewczyk E, Nayak T, Oakley CE, et al. Fusion PCR and gene targeting in Aspergillus nidulans. Nat Protoc. 2006;1:3111-3120.   DOI
8 Peberdy JF. 1995. Fungal protoplasts. In: Kuck U, editor. Genetics and biotechnology. The mycota (a comprehensive treatise on fungi as experimental systems for basic and applied research). Berlin (Germany): Springer. p. 49-60.
9 de Groot MJ, Bundock P, Hooykaas PJ, et al. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol. 1998;16:839-842.   DOI
10 Park S-M. Improved transformation of the filamentous fungus Aspergillus niger using Agrobacterium tumefaciens. Mycobiology. 2001;29:132-134.   DOI
11 Sugui JA, Chang YC, Kwon-Chung KJ. Agrobacterium tumefaciens-mediated transformation of Aspergillus fumigatus: an efficient tool for insertional mutagenesis and targeted gene disruption. Appl Environ Microbiol. 2005;71:1798-1802.   DOI
12 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. J Microbiol Methods. 2017;135:26-34.   DOI
13 Gelvin SB. Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool. Microbiol Mol Biol Rev. 2003;67:16-37.   DOI
14 Kunitake E, Tani S, Sumitani J, et al. Agrobacterium tumefaciens-mediated transformation of Aspergillus aculeatus for insertional mutagenesis. AMB Express. 2011;1:46.   DOI
15 Sun Y, Niu Y, He B, et al. A dual selection marker transformation system using Agrobacterium tumefaciens for the industrial Aspergillus oryzae 3.042. J Microbiol Biotechnol. 2019;29:230-234.   DOI
16 Nguyen KT, Ho QN, Pham TH, et al. The construction and use of versatile binary vectors carrying pyrG auxotrophic marker and fluorescent reporter genes for Agrobacterium-mediated transformation of Aspergillus oryzae. World J Microbiol Biotechnol. 2016;32:204.   DOI
17 Nguyen KT, Ho QN, Do L, et al. A new and efficient approach for construction of uridine/uracil auxotrophic mutants in the filamentous fungus Aspergillus oryzae using Agrobacterium tumefaciens-mediated transformation. World J Microbiol Biotechnol. 2017;33:107.   DOI
18 Li M, Zhou L, Liu M, et al. Construction of an engineering strain producing high yields of a-transglucosidase via Agrobacterium tumefaciens-mediated transformation of Asperillus niger. Biosci Biotechnol Biochem. 2013;77:1860-1866.   DOI
19 Mora-Lugo R, Zimmermann J, Rizk AM, et al. Development of a transformation system for Aspergillus sojae based on the Agrobacterium tumefaciens-mediated approach. BMC Microbiol. 2014;14:247.   DOI
20 Wang D, He D, Li G, et al. An efficient tool for random insertional mutagenesis: Agrobacterium tumefaciens-mediated transformation of the filamentous fungus Aspergillus terreus. J Microbiol Methods. 2014;98:114-118.   DOI
21 Han G, Shao Q, Li C, et al. An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus. J Microbiol. 2018;56:356-364.   DOI
22 Perrone G, Gallo A. Aspergillus species and their associated mycotoxins. Methods Mol Biol. 2017;1542:33-49.   DOI
23 Bennett JW. An overview of the genus Aspergillus. In: Machida M, Gomi K, editors. Aspergillus: molecular biology and genomics. Norfolk (UK): Caister Academic Press; 2010. p. 1-17.
24 Latge JP. Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev. 1999;12:310-350.   DOI
25 Bastos RW, Valero C, Silva LP, et al. Functional characterization of clinical isolates of the opportunistic fungal pathogen Aspergillus nidulans. mSphere. 2020;5:e00153-20.
26 Park HS, Jun SC, Han KH, et al. Diversity, application, and synthetic biology of industrially important Aspergillus fungi. Adv Appl Microbiol. 2017;100:161-202.   DOI
27 Lu H, Cao W, Liu X, et al. Multi-omics integrative analysis with genome-scale metabolic model simulation reveals global cellular adaptation of Aspergillus niger under industrial enzyme production condition. Sci Rep. 2018;8:14404.   DOI
28 Song R, Zhai Q, Sun L, et al. CRISPR/Cas9 genome editing technology in filamentous fungi: progress and perspective. Appl Microbiol Biotechnol. 2019;103:6919-6932.   DOI
29 Balabanova LA, Shkryl YN, Slepchenko LV, et al. Development of host strains and vector system for an efficient genetic transformation of filamentous fungi. Plasmid. 2019;101:1-9.   DOI
30 Xie H, Ma Q, Wei D, et al. Metabolic engineering of an industrial Aspergillus niger strain for itaconic acid production. 3 Biotech. 2020;10:113.   DOI
31 Niu J, Arentshorst M, Seelinger F, et al. A set of isogenic auxotrophic strains for constructing multiple gene deletion mutants and parasexual crossings in Aspergillus niger. Arch Microbiol. 2016;198:861-868.   DOI
32 Takahashi T, Masuda T, Koyama Y. Enhanced gene targeting frequency in ku70 and ku80 disruption mutants of Aspergillus sojae and Aspergillus oryzae. Mol Genet Genomics. 2006;275:460-470.   DOI
33 He B, Tu Y, Jiang C, et al. Functional genomics of Aspergillus oryzae: strategies and progress. Microorganisms. 2019;7:103.   DOI
34 Ventura L, Ramon D. Transformation of Aspergillus terreus with the hygromycin B resistance marker from Escherichia coli. FEMS Microbiol Lett. 1991;66:189-193.   DOI
35 Maruyama J, Kitamoto K. Multiple gene disruptions by marker recycling with highly efficient gene-targeting background (DeltaligD) in Aspergillus oryzae. Biotechnol Lett. 2008;30:1811-1817.   DOI
36 Tani S, Tsuji A, Kunitake E, et al. Reversible impairment of the ku80 gene by a recyclable marker in Aspergillus aculeatus. AMB Express. 2013;3:4.   DOI
37 Samson RA, Visagie CM, Houbraken J, et al. Phylogeny, identification and nomenclature of the genus Aspergillus. Stud Mycol. 2014;78:141-173.   DOI
38 Li D, Tang Y, Lin J, et al. Methods for genetic transformation of filamentous fungi. Microb Cell Fact. 2017;16:168.   DOI
39 Herzog RW, Daniell H, Singh NK, et al. A comparative study on the transformation of Aspergillus nidulans by microprojectile bombardment of conidia and a more conventional procedure using protoplasts treated with polyethyleneglycol. Appl Microbiol Biotechnol. 1996;45:333-337.   DOI
40 He ZM, Price MS, Obrian GR, et al. Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus. BMC Microbiol. 2007;7:104.   DOI
41 de Vries RP, Riley R, Wiebenga A, et al. Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus. Genome Biol. 2017;18:28.   DOI
42 Paulussen C, Hallsworth JE, Alvarez-Perez S, et al. Ecology of aspergillosis: insights into the pathogenic potency of Aspergillus fumigatus and some other Aspergillus species. Microb Biotechnol. 2017;10:296-322.   DOI
43 Latge JP, Chamilos G. Aspergillus fumigatus and Aspergillosis in 2019. Clin Microbiol Rev. 2019; 33:e00140-18.
44 Hedayati MT, Pasqualotto AC, Warn PA, et al. Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology (Reading). 2007;153:1677-1692.   DOI
45 Perrone G, Susca A, Cozzi G, et al. Biodiversity of Aspergillus species in some important agricultural products. Stud Mycol. 2007;59:53-66.   DOI
46 Bourdichon F, Casaregola S, Farrokh C, et al. Food fermentations: microorganisms with technological beneficial use. Int J Food Microbiol. 2012;154:87-97.   DOI
47 Agriopoulou S, Stamatelopoulou E, Varzakas T. Advances in analysis and detection of major mycotoxins in foods. Foods. 2020;9:518.   DOI
48 Kitamoto K. Cell biology of the Koji mold Aspergillus oryzae. Biosci Biotechnol Biochem. 2015;79:863-869.   DOI
49 Cairns TC, Nai C, Meyer V. How a fungus shapes biotechnology: 100 years of Aspergillus niger research. Fungal Biol Biotechnol. 2018;5:13.   DOI
50 Ojeda-Lopez M, Chen W, Eagle CE, et al. Evolution of asexual and sexual reproduction in the aspergilli. Stud Mycol. 2018;91:37-59.   DOI
51 Wang S, Chen H, Tang X, et al. Molecular tools for gene manipulation in filamentous fungi. Appl Microbiol Biotechnol. 2017;101:8063-8075.   DOI
52 Yoon J, Maruyama J, Kitamoto K. Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins. Appl Microbiol Biotechnol. 2011;89:747-759.   DOI
53 Nodvig CS, Nielsen JB, Kogle ME, et al. A CRISPR-Cas9 system for genetic engineering of filamentous fungi. PLoS One. 2015;10:e0133085.   DOI
54 Ruiz-Diez B. Strategies for the transformation of filamentous fungi. J Appl Microbiol. 2002;92:189-195.   DOI
55 van den Hombergh JP, van de Vondervoort PJ, Fraissinet-Tachet L, et al. Aspergillus as a host for heterologous protein production: the problem of proteases. Trends Biotechnol. 1997;15:256-263.   DOI
56 Jin FJ, Maruyama J, Juvvadi PR, et al. Development of a novel quadruple auxotrophic host transformation system by argB gene disruption using adeA gene and exploiting adenine auxotrophy in Aspergillus oryzae. FEMS Microbiol Lett. 2004;239:79-85.   DOI
57 Dohn JW Jr, Grubbs AW, Oakley CE, et al. New multi-marker strains and complementing genes for Aspergillus nidulans molecular biology. Fungal Genet Biol. 2018;111:1-6.   DOI
58 Fan Z, Yu H, Guo Q, et al. Identification and characterization of an anti-oxidative stress-associated mutant of Aspergillus fumigatus transformed by Agrobacterium tumefaciens. Mol Med Rep. 2016;13:2367-2376.   DOI
59 Chang PK, Scharfenstein LL, Wei Q, et al. Development and refinement of a high-efficiency gene-targeting system for Aspergillus flavus. J Microbiol Methods. 2010;81:240-246.   DOI
60 Pronk JT. Auxotrophic yeast strains in fundamental and applied research. Appl Environ Microbiol. 2002;68:2095-2100.   DOI
61 Matsuda Y, Bai T, Phippen CBW, et al. Novofumigatonin biosynthesis involves a non-heme iron-dependent endoperoxide isomerase for orthoester formation. Nat Commun. 2018;9:2587.   DOI
62 Barrangou R, Marraffini LA. CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity. Mol Cell. 2014;54:234-244.   DOI
63 Chakraborty BN, Kapoor M. Transformation of filamentous fungi by electroporation. Nucleic Acids Res. 1990;18:6737.   DOI
64 Ozeki K, Kyoya F, Hizume K, et al. Transformation of intact Aspergillus niger by electroporation. Biosci Biotechnol Biochem. 1994;58:2224-2227.   DOI
65 Gaj T, Sirk SJ, Shui SL, et al. Genome-editing technologies: principles and applications. Cold Spring Harb Perspect Biol. 2016;8:a023754.   DOI
66 Rath D, Amlinger L, Rath A, et al. The CRISPR-Cas immune system: biology, mechanisms and applications. Biochimie. 2015;117:119-128.   DOI
67 Fuller KK, Chen S, Loros JJ, et al. Development of the CRISPR/Cas9 system for targeted gene disruption in Aspergillus fumigatus. Eukaryot Cell. 2015;14:1073-1080.   DOI
68 Zheng X, Zheng P, Zhang K, et al. 5S rRNA promoter for guide RNA expression enabled highly efficient CRISPR/Cas9 genome editing in Aspergillus niger. ACS Synth Biol. 2019;8:1568-1574.   DOI
69 Leynaud-Kieffer LMC, Curran SC, Kim I, et al. A new approach to Cas9-based genome editing in Aspergillus niger that is precise, efficient and selectable. PLoS One. 2019;14:e0210243.   DOI
70 Kadooka C, Yamaguchi M, Okutsu K, et al. A CRISPR/Cas9-mediated gene knockout system in Aspergillus luchuensis mut. kawachii. Biosci Biotechnol Biochem. 2020;84:2179-2183.   DOI
71 Al Abdallah Q, Ge W, Fortwendel JR. A simple and universal system for gene manipulation in Aspergillus fumigatus: in vitro-assembled Cas9-guide RNA ribonucleoproteins coupled with microhomology repair templates. mSphere. 2017;2:e00446-17.
72 Palmer LM, Cove DJ. Pyrimidine biosynthesis in Aspergillus nidulans: isolation and preliminary characterisation of auxotrophic mutants. Mol Gen Genet. 1975;138:243-255.   DOI
73 Xue T, Nguyen CK, Romans A, et al. Isogenic auxotrophic mutant strains in the Aspergillus fumigatus genome reference strain AF293. Arch Microbiol. 2004;182:346-353.   DOI
74 da Silva Ferreira ME, Kress MR, Savoldi M, et al. The akuB(KU80) mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryot Cell. 2006;5:207-211.   DOI
75 Nayak T, Szewczyk E, Oakley CE, et al. A versatile and efficient gene-targeting system for Aspergillus nidulans. Genetics. 2006;172:1557-1566.   DOI
76 Nakamura H, Katayama T, Okabe T, et al. Highly efficient gene targeting in Aspergillus oryzae industrial strains under ligD mutation introduced by genome editing: strain-specific differences in the effects of deleting EcdR, the negative regulator of sclerotia formation. J Gen Appl Microbiol. 2017;63:172-178.   DOI
77 Zhang C, Meng X, Wei X, et al. Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus. Fungal Genet Biol. 2016;86:47-57.   DOI
78 Katayama T, Tanaka Y, Okabe T, et al. Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae. Biotechnol Lett. 2016;38:637-642.   DOI
79 Weber J, Valiante V, Nodvig CS, et al. Functional reconstitution of a fungal natural product gene cluster by advanced genome editing. ACS Synth Biol. 2017;6:62-68.   DOI
80 Nodvig CS, Hoof JB, Kogle ME, et al. Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli. Fungal Genet Biol. 2018;115:78-89.   DOI
81 Yu JH, Hamari Z, Han KH, et al. Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol. 2004;41:973-981.   DOI
82 Suzuki S, Tada S, Fukuoka M, et al. A novel transformation system using a bleomycin resistance marker with chemosensitizers for Aspergillus oryzae. Biochem Biophys Res Commun. 2009;383:42-47.   DOI
83 Meyer V, Arentshorst M, El-Ghezal A, et al. Highly efficient gene targeting in the Aspergillus niger kusA mutant. J Biotechnol. 2007;128:770-775.   DOI
84 Gravelat FN, Askew DS, Sheppard DC. Targeted gene deletion in Aspergillus fumigatus using the hygromycin-resistance split-marker approach. Methods Mol Biol. 2012;845:119-130.   DOI
85 Punt PJ, Oliver RP, Dingemanse MA, et al. Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene. 1987;56:117-124.   DOI
86 Oakley BR, Rinehart JE, Mitchell BL, et al. Cloning, mapping and molecular analysis of the pyrG (orotidine-5'-phosphate decarboxylase) gene of Aspergillus nidulans. Gene. 1987;61:385-399.   DOI
87 Nielsen ML, Albertsen L, Lettier G, et al. Efficient PCR-based gene targeting with a recyclable marker for Aspergillus nidulans. Fungal Genet Biol. 2006;43:54-64.   DOI
88 Setoguchi S, Mizutani O, Yamada O, et al. Effect of pepA deletion and overexpression in Aspergillus luchuensis on sweet potato shochu brewing. J Biosci Bioeng. 2019;128:456-462.   DOI
89 Sheppard DC, Doedt T, Chiang LY, et al. The Aspergillus fumigatus StuA protein governs the up-regulation of a discrete transcriptional program during the acquisition of developmental competence. Mol Biol Cell. 2005;16:5866-5879.   DOI
90 Min T, Xiong L, Liang Y, et al. Disruption of stcA blocks sterigmatocystin biosynthesis and improves echinocandin B production in Aspergillus delacroxii. World J Microbiol Biotechnol. 2019;35:109.   DOI
91 Kalleda N, Naorem A, Manchikatla RV. Targeting fungal genes by diced siRNAs: a rapid tool to decipher gene function in Aspergillus nidulans. PLoS One. 2013;8:e75443.   DOI
92 Kuck U, Hoff B. New tools for the genetic manipulation of filamentous fungi. Appl Microbiol Biotechnol. 2010;86:51-62.   DOI
93 Meyer V, Mueller D, Strowig T, et al. Comparison of different transformation methods for Aspergillus giganteus. Curr Genet. 2003;43:371-377.   DOI
94 Zhao C, Fraczek MG, Dineen L, et al. High-throughput gene replacement in Aspergillus fumigatus. Curr Protoc Microbiol. 2019;54:e88.
95 Gouka RJ, Gerk C, Hooykaas PJ, et al. Transformation of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologous recombination. Nat Biotechnol. 1999;17:598-601.   DOI
96 Michielse CB, Hooykaas PJ, van den Hondel CA, et al. Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori. Nat Protoc. 2008;3:1671-1678.   DOI
97 Chakraborty BN, Patterson NA, Kapoor M. An electroporation-based system for high-efficiency transformation of germinated conidia of filamentous fungi. Can J Microbiol. 1991;37:858-863.   DOI
98 Weidner G, d'Enfert C, Koch A, et al. Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5'-monophosphate decarboxylase. Curr Genet. 1998;33:378-385.   DOI
99 Zhu SY, Xu Y, Yu XW. Improved homologous expression of the acidic lipase from Aspergillus niger. J Microbiol Biotechnol. 2020;30:196-205.   DOI
100 Richey MG, Marek ET, Schardl CL, et al. Transformation of filamentous fungi with plasmid DNA by electroporation. Phytopathology. 1989;79:844-847.   DOI
101 Sanchez O, Aguirre J. Efficient transformation of Aspergillus nidulans by electroporation of germinated conidia. Fungal Genet Newsl. 1996;43: 48-51.
102 Brown JS, Aufauvre-Brown A, Holden DW. Insertional mutagenesis of Aspergillus fumigatus. Mol Gen Genet. 1998;259:327-335.   DOI
103 Firon A, Beauvais A, Latge JP, et al. Characterization of essential genes by parasexual genetics in the human fungal pathogen Aspergillus fumigatus: impact of genomic rearrangements associated with electroporation of DNA. Genetics. 2002;161:1077-1087.   DOI
104 Firon A, Villalba F, Beffa R, et al. Identification of essential genes in the human fungal pathogen Aspergillus fumigatus by transposon mutagenesis. Eukaryot Cell. 2003;2:247-255.   DOI