Identification and characterization of the ZRT, IRT-like protein (ZIP) family genes reveal their involvement in growth and kojic acid production in Aspergillus oryzae
- 208 Downloads
The ZRT, IRT-like protein (ZIP) family exists in many species and plays an important role in many biological processes, but little is known about ZIP genes in Aspergillus oryzae. Here, 10 ZIP genes in A. oryzae were identified and these were classified into four groups based on phylogenetic analysis. The structures of these AoZip genes were determined, which indicated a great divergence of AoZip members from different groups. Synteny analysis revealed that AoZip7, AoZip8, and AoZip10 are conserved among Aspergillus species. We also found that the promoter regions of AoZip2, AoZip7, AoZip8, and AoZip10 contain multiple conserved response elements. Expression analysis revealed that AoZips exhibited different expression patterns in response to different metal treatments. Moreover, overexpression and RNA-interference (RNAi) of AoZip2 led to a decrease in mycelium growth diameter and inhibited conidia formation. AoZip2 overexpression and RNAi strains showed distinct sensitivity to severely Zn/Mn-depleted stress. In addition, kojic acid production was markedly lower in AoZip2 overexpression and RNAi strains than in the control strains, and the expression of kojA, kojR, and kojT was down-regulated in AoZip2 overexpression and RNAi strains. This study provides new insights into our understanding of ZIP genes and lays a foundation for further investigation of their roles in Aspergillus oryzae.
KeywordsAspergillus oryzae ZIP transporter Kojic acid Secondary metabolism
This work was supported by the Natural Science Foundation of China (31460447 and 31171731) and Doctoral Scientific Research Foundation of Jiangxi Science and Technology Normal University (3000990632).
Compliance with ethical standards
Conflict of interest
All authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- 2.Amich J, Vicentefranqueira R, Leal F, Calera JA (2010) Aspergillus fumigatus survival in alkaline and extreme zinc-limiting environments relies on the induction of a zinc homeostasis system encoded by the zrfC and aspf2 genes. Eukaryot Cell 9:424–437. https://doi.org/10.1128/EC.00348-09 CrossRefPubMedPubMedCentralGoogle Scholar
- 3.Amich J, Vicentefranqueira R, Mellado E, Ruiz-Carmuega A, Leal F, Calera JA (2014) The ZrfC alkaline zinc transporter is required for Aspergillus fumigatus virulence and its growth in the presence of the Zn/Mn-chelating protein calprotectin. Cell Microbiol 16:548–564. https://doi.org/10.1111/cmi.12238 CrossRefPubMedGoogle Scholar
- 4.Arakawa GY, Kudo H, Yanase A, Eguchi Y, Kodama H, Ogawa M, Koyama Y, Shindo H, Hosaka M, Tokuoka M (2019) A unique Zn(II)2-Cys6-type protein, KpeA, is involved in secondary metabolism and conidiation in Aspergillus oryzae. Fungal Genet Biol 127:35–44. https://doi.org/10.1016/j.fgb.2019.02.004 CrossRefPubMedGoogle Scholar
- 6.Blatzer M, Barker BM, Willger SD, Beckmann N, Blosser SJ, Cornish EJ, Mazurie A, Grahl N, Haas H, Cramer RA (2011) SREBP coordinates iron and ergosterol homeostasis to mediate triazole drug and hypoxia responses in the human fungal pathogen Aspergillus fumigatus. PLoS Genet 7:e1002374. https://doi.org/10.1371/journal.pgen.1002374 CrossRefPubMedPubMedCentralGoogle Scholar
- 7.Christensen T, Hynes MJ, Davis MA (1998) Role of the regulatory gene areA of Aspergillus oryzae in nitrogen metabolism. Appl Environ Biotechnol 64:3232–3237Google Scholar
- 26.Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai G, Kusumoto K, Arima T, Akita O, Kashiwagi Y, Abe K, Gomi K, Horiuchi H, Kitamoto K, Kobayashi T, Takeuchi M, Denning DW, Galagan JE, Nierman WC, Yu J, Archer DB, Bennett JW, Bhatnagar D, Cleveland TE, Fedorova ND, Gotoh O, Horikawa H, Hosoyama A, Ichinomiya M, Igarashi R, Iwashita K, Juvvadi PR, Kato M, Kato Y, Kin T, Kokubun A, Maeda H, Maeyama N, Maruyama J, Nagasaki H, Nakajima T, Oda K, Okada K, Paulsen I, Sakamoto K, Sawano T, Takahashi M, Takase K, Terabayashi Y, Wortman JR, Yamada O, Yamagata Y, Anazawa H, Hata Y, Koide Y, Komori T, Koyama Y, Minetoki T, Suharnan S, Tanaka A, Isono K, Kuhara S, Ogasawara N, Kikuchi H (2005) Genome sequencing and analysis of Aspergillus oryzae. Nature 438:1157–1161. https://doi.org/10.1038/nature04300 CrossRefPubMedGoogle Scholar
- 28.Marui J, Yamane N, Ohashi-Kunihiro S, Ando T, Terabayashi Y, Sano M, Ohashi S, Ohshima E, Tachibana K, Higa Y, Nishimura M, Koike H, Machida M (2011) Kojic acid biosynthesis in Aspergillus oryzae is regulated by a Zn(II)2Cys6 transcriptional activator and induced by kojic acid at the transcriptional level. J Biosci Bioeng 112:40–43. https://doi.org/10.1016/j.jbiosc.2011.03.010 CrossRefPubMedGoogle Scholar
- 36.Schrettl M, Kim HS, Eisendle M, Kragl C, Nierman WC, Heinekamp T, Werner ER, Jacobsen I, Illmer P, Yi H, Brakhage AA, Haas H (2008) SreA-mediated iron regulation in Aspergillus fumigatus. Mol Microbiol 70:27–43. https://doi.org/10.1111/j.1365-2958.2008.06376.x CrossRefPubMedPubMedCentralGoogle Scholar
- 38.Terabayashi Y, Sano M, Yamane N, Marui J, Tamano K, Sagara J, Dohmoto M, Oda K, Ohshima E, Tachibana K, Higa Y, Ohashi S, Koike H, Machida M (2010) Identification and characterization of genes responsible for biosynthesis of kojic acid, an industrially important compound from Aspergillus oryzae. Fungal Genet Biol 47:953–961. https://doi.org/10.1016/j.fgb.2010.08.014 CrossRefPubMedGoogle Scholar
- 39.Vicentefranqueira R, Moreno MA, Leal F, Calera JA (2005) The zrfA and zrfB genes of Aspergillus fumigatus encode the zinc transporter proteins of a zinc uptake system induced in an acid, zinc-depleted environment. Eukaryot Cell 4:837–848. https://doi.org/10.1128/EC.4.5.837-848.2005 CrossRefPubMedPubMedCentralGoogle Scholar
- 41.Yamada R, Yoshie T, Wakai S, Asai-Nakashima N, Okazaki F, Ogino C, Hisada H, Tsutsumi H, Hata Y, Kondo A (2014) Aspergillus oryzae-based cell factory for direct kojic acid production from cellulose. Microb Cell Fact 13:71. https://doi.org/10.1186/1475-2859-13-71 CrossRefPubMedPubMedCentralGoogle Scholar