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Construction and characterization of the GFAT gene as a novel selection marker in Aspergillus nidulans

  • Applied genetics and molecular biotechnology
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Abstract

Glutamine:fructose-6-phosphate aminotransferase (GFAT) catalyzes the formation of glucosamine-6-phosphate, and its gene is one of the genes essential for microbes. Using the GFAT-encoding gene can prevent the use of a drug-resistant gene as a selection marker in a bacterial system. Another unique property of the GFAT selection marker is that no particular compound is prohibited or required for creating a selective stress for a yeast. Filamentous fungi are major producers of industrial enzymes. However, there has been no report on the construction and application of the GFAT gene as a selection marker in filamentous fungi. To develop a new selection marker, the GFAT-encoding gene gfaA was deleted from the genome of the filamentous fungus Aspergillus nidulans, and the gfat gene of the straw mushroom Volvariella volvacea was used as the selection marker to mediate the transformation and overexpression of a thermostable bacterial laccase in A. nidulans. The GFAT-deficient strain A. nidulans ∆gfaA was not able to grow in the culture medium containing 0.5% yeast extract unless about 20 mM glucosamine was used to supplement to the medium. The gfat gene was amplified and inserted into the integration vector pAL5 and autonomous replication vector Prg3-AMA1-NotI for A. nidulans to generate the gfat vectors pALG and pAMAG, respectively. Using these gfat vectors, the laccase gene lcs from a hyperthermophilic bacterium was overexpressed intra- and extracellularly in A. nidulans ∆gfaA. Therefore, recombinant filamentous fungi can be constructed with gfat vectors, which can be maintained stably in host cells with the naturally occurred selective stress of a medium, forage, pulp, animal gut, wastewater, or soil.

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References

  • Adams TH, Wieser JK, Yu JH (1998) Asexual sporulation in Aspergillus nidulans. Microbiol Mol Biol Rev 62:35–54

    PubMed  PubMed Central  CAS  Google Scholar 

  • Aleksenko AY, Clutterbuck AJ (1995) Recombinational stability of replicating plasmids in Aspergillus nidulans during transformation, vegetative growth and sexual reproduction. Curr Genet 28:87–93

    Article  PubMed  CAS  Google Scholar 

  • Aleksenko A, Nikolaev I, Vinetski Y, Clutterbuck AJ (1996) Gene expression from replicating plasmids in Aspergillus nidulans. Mol Gen Genet 253:242–246

    Article  PubMed  CAS  Google Scholar 

  • Andersson DI, Levin BR (1999) The biological cost of antibiotic resistance. Curr Opin Microbiol 2:489–493

    Article  PubMed  CAS  Google Scholar 

  • Apolinario E, Nocero M, Jin M, Hoffman CS (1993) Cloning and manipulation of the Schizosaccharomyces pombe his7 + gene as a new selection marker for molecular genetic studies. Curr Genet 24:491–495

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Armitt S, McCullough W, Roberts CF (1976) Analysis of acetate non-utilizing (acu) mutants in Aspergillus nidulans. J Gen Microbiol 92:263–282

    Article  PubMed  CAS  Google Scholar 

  • Azizi M, Yakhchali B, Ghamarian A, Enayati S, Khodabandeh M, Khalaj V (2013) Cloning and expression of gumboro VP2 antigen in Aspergillus niger. Avicenna J Med Biotechnol 5:35–41

    PubMed  PubMed Central  CAS  Google Scholar 

  • Blin N, Stafford DW (1976) A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res 3:2303–2308

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Burke JD, Gould KL (1994) Molecular cloning and characterization of the Schizosaccharomyces pombe his3 gene for use as a selection marker. Mol Gen Genet 242:169–176

    Article  PubMed  CAS  Google Scholar 

  • Buxton FP, Gwynne DI, Davies RW (1989) Cloning of a new bidirectionally selection marker for Aspergillus strains. Gene 84:329–334

    Article  PubMed  CAS  Google Scholar 

  • Clutterbuck AJ (1972) Absence of laccase from yellow-spored mutants of Aspergillus nidulans. J Gen Microbiol 70:423–435

    Article  PubMed  CAS  Google Scholar 

  • Cullen D, Gray GL, Wilson LJ, Hayenga KJ, Lamsa MH, Rey MW, Norton S, Berka RM (1987) Controlled expression and secretion of bovine chymosin in Aspergillus nidulans. Nat Biotechnol 5:369–376

    Article  CAS  Google Scholar 

  • Cove DJ (1966) The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim Biophys Acta 113:51–56

    Article  PubMed  CAS  Google Scholar 

  • Doonan JH, MacKintosh C, Osmani S, Cohen P, Bai G, Lee EY, Morris NR (1991) A cDNA encoding rabbit muscle protein phosphatase 1α complements the Aspergillus cell cycle mutation, bimG11. J Biol Chem 266:18889–18894

    PubMed  CAS  Google Scholar 

  • Dutka-Malen S, Mazodier P, Badet B (1988) Molecular cloning and overexpression of the glucosamine synthetase gene from Escherichia coli. Biochimie 70:287–290

    Article  PubMed  CAS  Google Scholar 

  • d’Enfert C (1996) Selection of multiple disruption events in Aspergillus fumigatus using the orotidine-5′-decarboxylase gene, pyrG, as a unique transformation marker. Curr Genet 30:76–82

    Article  PubMed  Google Scholar 

  • Gems D, Aleksenko A, Belenky L, Robertson S, Ramsden M, Vinetski Y, Clutterbuck AJ (1994) An 'instant gene bank' method for gene cloning by mutant complementation. Mol Gen Genet 242:467–471

    Article  PubMed  CAS  Google Scholar 

  • Germs D, Johnstone IL, Clutterbuck AJ (1991) An autonomously replicating plasmid transforms Aspergillus nidulans at high frequency. Gene 98:61–67

    Article  Google Scholar 

  • Hoffman RL, Hoffman CS (2006) Cloning the Schizosaccharomyces pombe lys2+ gene and construction of new molecular genetic tools. Curr Genet 49:414–420

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Käfer E (1977) Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations. Adv Genet 19:33–131

    PubMed  Google Scholar 

  • Le Y, Peng J, Wu H, Sun J, Shao W (2011) An approach for production of soluble protein from a fungal gene encoding an aggregation-prone xylanase in Escherichia coli. PLoS One 6:e18489

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Liu B, Morris NR (2000) A spindle pole body-associated protein, SNAD, affects septation and conidiation in Aspergillus nidulans. Mol Gen Genet 263:375–387

    Article  PubMed  CAS  Google Scholar 

  • Luo C, Shao W, Li X, Chen Z, Liu Y (2009) Molecular cloning, sequencing, and expression of a L-glutamine D-fructose 6-phosphate amidotransferase gene from Volvariella volvacea. Protein J 28:34–43

    Article  PubMed  CAS  Google Scholar 

  • Lubertozzi D, Keasling JD (2009) Developing Aspergillus as host for heterologous expression. Biotechnol Adv 27:53–75

    Article  PubMed  CAS  Google Scholar 

  • Mander GJ, Wang H, Bodie E, Wagner J, Vienken K, Vinuesa C, Foster C, Leeder AC, Allen G, Hamill V, Janssen GG, Dunn-Coleman N, Karos M, Lemaire HG, Subkowski T, Bollschweiler C, Turner G, Nüsslein B, Fischer R (2006) Use of laccase as a novel, versatile reporter system in filamentous fungi. Appl Environ Microbiol 72:5020–5026

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Moreno MA, Pascual C, Gibello A, Ferrer S, Bos CJ, Debets AJM, Suárez G (1994) Transformation of Aspergillus parasiticus using autonomously replicating plasmids from Aspergillus nidulans. FEMS Microbiol Lett 124:35–42

    Article  PubMed  CAS  Google Scholar 

  • Nayak T, Szewczyk E, Oakley CE, Osmani A, Ukil L, Murray SL, Hynes MJ, Osmani SA, Oakley BR (2006) A versatile and efficient gene-targeting system for Aspergillus nidulans. Genetics 172:1557–1566

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • O’Gorman CM, Fuller HT, Dyer PS (2009) Discovery of a sexual cycle in the opportunistic fungal pathogen Aspergillus fumigatus. Nature 457:471–474

    Article  PubMed  CAS  Google Scholar 

  • Osherov N, Mathew J, May GS (2000) Polarity-defective mutants of Aspergillus nidulans. Fungal Genet Biol 31:181–188

    Article  PubMed  CAS  Google Scholar 

  • Osmani SA, Pu RT, Morris NR (1988) Mitotic induction and maintenance by overexpression of a G2-specific gene that encodes a potential protein-kinase. Cell 53:237–244

    Article  PubMed  CAS  Google Scholar 

  • Punt PJ, Dingemanse MA, Kuyvenhoven A, Soede RDM, Pouwels PH, van den Hondel CA (1990) Functional elements in the promoter region of the Aspergillus nidulans gpdA gene coding for glyceraldehyde-3-phosphate dehydrogenase. Gene 93:101–109

    Article  PubMed  CAS  Google Scholar 

  • Ram AF, Arentshorst M, Damveld RA, vanKuyk PA, Klis FM, van den Hondel CA (2004) The cell wall stress response in Aspergillus niger involves increased expression of the glutamine: D-fructose-6-phosphate amidotransferase-encoding gene (gfaA) and increased deposition of chitin in the cell wall. Microbiology 150:3315–3326

    Article  PubMed  CAS  Google Scholar 

  • Ratledge C, Kristiansen B (2001) Public perception of biotechnology. In: In (ed) Smith JE (ed)Basic biotechnology. Cambridge University Press, Cambridge, pp 1–12

    Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning a laboratory manual. Cold Spring Harbour Laboratory Press, New York

    Google Scholar 

  • Shao WL, Wang HC (2013) Construction and application of yeast-bacterial shuttle vector without drug-resistant selection marker genes. Chinese Patent ZL 201310191146:4

    Google Scholar 

  • Verdoes JC, Punt PJ, van den Berg P, Debets F, Stouthamer A, van den Hondel CA (1994) Characterization of an efficient gene cloning strategy for Aspergillus niger based on an autonomously replicating plasmid: cloning of the nicB gene of A. niger. Gene 146:159–165

    Article  PubMed  CAS  Google Scholar 

  • Wu G, Sun Y, Qu W, Huang Y, Lu L, Li L, Shao W (2011) Application of GFAT as a novel selection marker to mediate gene expression. PLoS One 6:e17082

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Yamashiro CT, Yarden O, Yanofsky C (1992) A dominant selection marker that is meiotically stable in Neurospora crassa: the amdS gene of Aspergillus nidulans. Mol Gen Genet 236:121–124

    PubMed  CAS  Google Scholar 

  • Yelton MM, Hamer JE, Timberlake WE (1984) Transformation of Aspergillus nidulans by using a trpC plasmid. Proc Natl Acad Sci U S A 81:1470–1474

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Yu JH, Hamari Z, Han KH, Seo JA, Reyes-Domínguez Y, Scazzocchio C (2004) Double-joint PCR: a PCR-BASE molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol 41:973–981

    Article  PubMed  CAS  Google Scholar 

  • Zheng Z, Li H, Li L, Shao W (2012) Biobleaching of wheat straw pulp with recombinant laccase from the hyperthermophilic Thermus thermophilus HB27. Biotechnol Lett 34:541–547

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by a grant from the National Natural Science Foundation of China (grant nos. 30970062 and 31770089) and the Key Project of Science and Technology Program of Jiangsu Province, China (grant no. BE2016353).

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Authors and Affiliations

  1. College of Life and Science, Nanjing Normal University, Nanjing, 210023, JS, China

    Yu Sun, Hongcheng Wang & Weilan Shao

  2. School of Environment and Safety Engineering, Jiangsu University, 1 Xuefu Road, Zhenjiang, 212013, JS, China

    Hongcheng Wang & Weilan Shao

  3. Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, ON, N2L 3G1, Canada

    Kesen Ma

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  1. Yu Sun
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  2. Hongcheng Wang
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Correspondence to Kesen Ma or Weilan Shao.

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Sun, Y., Wang, H., Ma, K. et al. Construction and characterization of the GFAT gene as a novel selection marker in Aspergillus nidulans. Appl Microbiol Biotechnol 102, 7951–7962 (2018). https://doi.org/10.1007/s00253-018-9185-0

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