A New and Efficient Approach for Construction of Uridine/uracil Auxotrophic Mutants in the Filamentous Fungus Aspergillus Oryzae Using Agrobacterium Tumefaciens-mediated Transformation

Overview

Journal World J Microbiol Biotechnol

Publisher Springer

Specialties Biotechnology
Microbiology

Date 2017 May 4

PMID 28466303

Citations 22

Authors

Khuyen Thi Nguyen

Quynh Ngoc Ho

Loc Thi Binh Xuan Do

Linh Thi Dam Mai

Duc-Ngoc Pham

Huyen Thi Thanh Tran

Diep Hong Le

Huy Quang Nguyen

Van-Tuan Tran

Affiliations

Soon will be listed here.

Abstract

Aspergillus oryzae is a filamentous fungus widely used in food industry and as a microbial cell factory for recombinant protein production. Due to the inherent resistance of A. oryzae to common antifungal compounds, genetic transformation of this mold usually requires auxotrophic mutants. In this study, we show that Agrobacterium tumefaciens-mediated transformation (ATMT) method is very efficient for deletion of the pyrG gene in different Aspergillus oryzae wild-type strains to generate uridine/uracil auxotrophic mutants. Our data indicated that all the obtained uridine/uracil auxotrophic transformants, which are 5- fluoroorotic acid (5-FOA) resistant, exist as the pyrG deletion mutants. Using these auxotrophic mutants and the pyrG selectable marker for genetic transformation via A. tumefaciens, we could get about 1060 transformants per 10 fungal spores. In addition, these A. oryzae mutants were also used successfully for expression of the DsRed fluorescent reporter gene under control of the A. oryzae amyB promoter by the ATMT method, which resulted in obvious red transformants on agar plates. Our work provides a new and effective approach for constructing the uridine/uracil auxotrophic mutants in the importantly industrial fungus A. oryzae. This strategy appears to be applicable to other filamentous fungi to develop similar genetic transformation systems based on auxotrophic/nutritional markers for food-grade recombinant applications.

Citing Articles

Screening of pathogenicity-deficient Penicillium italicum mutants established by Agrobacterium tumefaciens-mediated transformation.

Zhang M, Yang S, Li Q, Wang M, Peng L Mol Genet Genomics. 2024; 299(1):82.

PMID: 39196386 DOI: 10.1007/s00438-024-02171-0.

Sub-genomic RNAi-assisted strain evolution of filamentous fungi for enhanced protein production.

Sun X, Gao F, Fan C, Yang S, Zhao T, Tu T Appl Environ Microbiol. 2024; 90(7):e0208223.

PMID: 38899886 PMC: 11267940. DOI: 10.1128/aem.02082-23.

The ancient koji mold (Aspergillus oryzae) as a modern biotechnological tool.

Daba G, Mostafa F, Elkhateeb W Bioresour Bioprocess. 2024; 8(1):52.

PMID: 38650252 PMC: 10992763. DOI: 10.1186/s40643-021-00408-z.

Construction of a New -Mediated Transformation System based on a Dual Auxotrophic Approach in .

Yan H, Shang Y, Wang L, Tian X, Tran V, Yao L J Microbiol Biotechnol. 2024; 34(5):1178-1187.

PMID: 38563100 PMC: 11180907. DOI: 10.4014/jmb.2312.12003.

Synthetic Biology Tools for Engineering .

Yang H, Song C, Liu C, Wang P J Fungi (Basel). 2024; 10(1).

PMID: 38248944 PMC: 10817548. DOI: 10.3390/jof10010034.

References

Zhong Y, Yu H, Wang X, Lu Y, Wang T . Towards a novel efficient T-DNA-based mutagenesis and screening system using green fluorescent protein as a vital reporter in the industrially important fungus Trichoderma reesei. Mol Biol Rep. 2010; 38(6):4145-51. DOI: 10.1007/s11033-010-0534-z. View

Zhu Y, Tramper J . Koji--where East meets West in fermentation. Biotechnol Adv. 2013; 31(8):1448-57. DOI: 10.1016/j.biotechadv.2013.07.001. View

MATTERN I, Unkles S, Kinghorn J, Pouwels P, van den Hondel C . Transformation of Aspergillus oryzae using the A. niger pyrG gene. Mol Gen Genet. 1987; 210(3):460-1. DOI: 10.1007/BF00327197. View

Michielse C, Arentshorst M, Ram A, van den Hondel C . Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori. Fungal Genet Biol. 2004; 42(1):9-19. DOI: 10.1016/j.fgb.2004.06.009. View

Michielse C, Hooykaas P, Van Den Hondel C, Ram A . Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr Genet. 2005; 48(1):1-17. DOI: 10.1007/s00294-005-0578-0. View

Lee L, Gelvin S . T-DNA binary vectors and systems. Plant Physiol. 2008; 146(2):325-32. PMC: 2245830. DOI: 10.1104/pp.107.113001. View

Kunitake E, Tani S, Sumitani J, Kawaguchi T . Agrobacterium tumefaciens-mediated transformation of Aspergillus aculeatus for insertional mutagenesis. AMB Express. 2011; 1(1):46. PMC: 3292464. DOI: 10.1186/2191-0855-1-46. View

Broekhuijsen M, Unkles S, CAMPBELL E, Kinghorn J, Contreras R, Pouwels P . A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae. Curr Genet. 1989; 16(3):159-63. DOI: 10.1007/BF00391472. View

Talhinhas P, Muthumeenakshi S, Neves-Martins J, Oliveira H, Sreenivasaprasad S . Agrobacterium-mediated transformation and insertional mutagenesis in Colletotrichum acutatum for investigating varied pathogenicity lifestyles. Mol Biotechnol. 2008; 39(1):57-67. DOI: 10.1007/s12033-007-9028-1. View

10.

Oakley B, Rinehart J, Mitchell B, Oakley C, Carmona C, Gray G . Cloning, mapping and molecular analysis of the pyrG (orotidine-5'-phosphate decarboxylase) gene of Aspergillus nidulans. Gene. 1987; 61(3):385-99. DOI: 10.1016/0378-1119(87)90201-0. View

11.

Tran V, Braus-Stromeyer S, Kusch H, Reusche M, Kaever A, Kuhn A . Verticillium transcription activator of adhesion Vta2 suppresses microsclerotia formation and is required for systemic infection of plant roots. New Phytol. 2014; 202(2):565-581. DOI: 10.1111/nph.12671. View

12.

Zhu L, Maruyama J, Kitamoto K . Further enhanced production of heterologous proteins by double-gene disruption (ΔAosedD ΔAovps10) in a hyper-producing mutant of Aspergillus oryzae. Appl Microbiol Biotechnol. 2013; 97(14):6347-57. DOI: 10.1007/s00253-013-4795-z. View

13.

Machida M, Yamada O, Gomi K . Genomics of Aspergillus oryzae: learning from the history of Koji mold and exploration of its future. DNA Res. 2008; 15(4):173-83. PMC: 2575883. DOI: 10.1093/dnares/dsn020. View

14.

van Hartingsveldt W, MATTERN I, van Zeijl C, Pouwels P, van den Hondel C . Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Mol Gen Genet. 1987; 206(1):71-5. DOI: 10.1007/BF00326538. View

15.

dEnfert C . Selection of multiple disruption events in Aspergillus fumigatus using the orotidine-5'-decarboxylase gene, pyrG, as a unique transformation marker. Curr Genet. 1996; 30(1):76-82. DOI: 10.1007/s002940050103. View

16.

de Groot M, Bundock P, Hooykaas P, Beijersbergen A . Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol. 1998; 16(9):839-42. DOI: 10.1038/nbt0998-839. View

17.

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S . MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013; 30(12):2725-9. PMC: 3840312. DOI: 10.1093/molbev/mst197. View

18.

Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai G . Genome sequencing and analysis of Aspergillus oryzae. Nature. 2005; 438(7071):1157-61. DOI: 10.1038/nature04300. View

19.

Nguyen K, Ho Q, Pham T, Phan T, Tran V . 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(12):204. DOI: 10.1007/s11274-016-2168-3. View

20.

Arentshorst M, Lagendijk E, Ram A . A new vector for efficient gene targeting to the locus in . Fungal Biol Biotechnol. 2017; 2:2. PMC: 5611571. DOI: 10.1186/s40694-015-0012-4. View