A new and efficient approach for construction of uridine/uracil auxotrophic mutants in the filamentous fungus Aspergillus oryzae using Agrobacterium tumefaciens-mediated transformation

Nguyen, Khuyen Thi; Ho, Quynh Ngoc; Xuan, Loc Thi Binh; Dam, Linh Thi; Pham, Duc-Ngoc; Tran, Huyen Thi Thanh; Le, Diep Hong; Nguyen, Huy Quang; Tran, Van‐Tuan

doi:10.1007/s11274-017-2275-9

Cited by 34 publications

(21 citation statements)

References 49 publications

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“…Since the first report of a DNA-mediated transformation for a fungus in 1973 [ 25 ], many fungal species, such as Sclerotinia sclerotiorum and Aspergillus oryzae , have been transformed not only for PEG-mediated transformation of protoplast, but also for Agrobacterium -mediated, biolistics, and electroporation transformation [ 26 , 27 , 28 ]. PEG-mediated protoplast transformation is an important method for studying gene function in filamentous fungi [ 13 ].…”

Section: Discussionmentioning

confidence: 99%

Optimization of Production Conditions for Protoplasts and Polyethylene Glycol-Mediated Transformation of Gaeumannomyces tritici

et al. 2018

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Take-all, caused by Gaeumannomyces tritici, is one of the most important wheat root diseases worldwide, as it results in serious yield losses. In this study, G. tritici was transformed to express the hygromycin B phosphotransferase using a combined protoplast and polyethylene glycol (PEG)-mediated transformation technique. Based on a series of single-factor experimental results, three major factors—temperature, enzyme lysis time, and concentration of the lysing enzyme—were selected as the independent variables, which were optimized using the response surface methodology. A higher protoplast yield of 9.83 × 107 protoplasts/mL was observed, and the protoplast vitality was also high, reaching 96.27% after optimization. Protoplasts were isolated under the optimal conditions, with the highest transformation frequency (46–54 transformants/μg DNA). Polymerase chain reaction and Southern blotting detection indicated that the genes of hygromycin phosphotransferase were successfully inserted into the genome of G. tritici. An optimised PEG-mediated protoplast transformation system for G. tritici was established. The techniques and procedures described will lay the foundation for establishing a good mutation library of G. tritici and could be used to transform other fungi.

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Section: Discussionmentioning

confidence: 99%

Optimization of Production Conditions for Protoplasts and Polyethylene Glycol-Mediated Transformation of Gaeumannomyces tritici

et al. 2018

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“…In yeast and filamentous fungi, the pyrG gene has been widely used as nutritional/auxotrophic marker for fungal genetic manipulation [21,22,[27][28][29][30]. With the advantage of counter selection using 5-FOA, pyrG could is utilized as the bidirectional selection marker for recyclable genome editing.…”

Section: Gene Disruption Of Pyrg and Pyrg/kusa Constructed By Crispr/mentioning

confidence: 99%

Disruption or reduced expression of the orotidine-5′-decarboxylase gene pyrG increases citric acid production: a new discovery during recyclable genome editing in Aspergillus niger

et al. 2020

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Background: Aspergillus niger is a filamentous fungus used for the majority of global citric acid production. Recent developments in genome editing now enable biotechnologists to engineer and optimize A. niger. Currently, however, genetic-leads for maximizing citric acid titers in industrial A. niger isolates is limited. Results: In this study, we try to engineer two citric acid A. niger production isolates, WT-D and D353, to serve as platform strains for future high-throughput genome engineering. Consequently, we used genome editing to simultaneously disrupt genes encoding the orotidine-5′-decarboxylase (pyrG) and non-homologous end-joining component (kusA) to enable use of the pyrG selection/counter selection system, and to elevate homologous recombination rates, respectively. During routine screening of these pyrG mutant strains, we unexpectedly observed a 2.17-fold increase in citric acid production when compared to the progenitor controls, indicating that inhibition of uridine/pyrimidine synthesis may increase citric acid titers. In order to further test this hypothesis, the pyrG gene was placed under the control of a tetracycline titratable cassette, which confirmed that reduced expression of this gene elevated citric acid titers in both shake flask and bioreactor fermentation. Subsequently, we conducted intracellular metabolomics analysis, which demonstrated that pyrG disruption enhanced the glycolysis flux and significantly improved abundance of citrate and its precursors. Conclusions: In this study, we deliver two citric acid producing isolates which are amenable to high throughput genetic manipulation due to pyrG/kusA deletion. Strikingly, we demonstrate for the first time that A. niger pyrG is a promising genetic lead for generating citric acid hyper-producing strains. Our data support the hypothesis that uridine/pyrimidine biosynthetic pathway offer future avenues for strain engineering efforts.

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“…Yasuda et al established an efficient gene knockout system using A. oryzae KBN630 as an original strain and pyrG as a selection marker [19]. Nguyen et al confirmed that the pyrG selectable marker is a powerful tool for genetic transformation and recombinant gene expression studies in A. oryzae [20]. Maruyama et al and Yoon et al knocked out multiple genes through a pyrG selectable marker [21,22].…”

Section: Strategies For Functional Genomics Of a Oryzaementioning

confidence: 99%

Functional Genomics of Aspergillus oryzae: Strategies and Progress

Ту

Jiang

et al. 2019

Microorganisms

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Aspergillus oryzae has been used for the production of traditional fermentation and has promising potential to produce primary and secondary metabolites. Due to the tough cell walls and high drug resistance of A. oryzae, functional genomic characterization studies are relatively limited. The exploitation of selection markers and genetic transformation methods are critical for improving A. oryzae fermentative strains. In this review, we describe the genome sequencing of various A. oryzae strains. Recently developed selection markers and transformation strategies are also described in detail, and the advantages and disadvantages of transformation methods are presented. Lastly, we introduce the recent progress on highlighted topics in A. oryzae functional genomics including conidiation, protein secretion and expression, and secondary metabolites, which will be beneficial for improving the application of A. oryzae to industrial production.

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A new and efficient approach for construction of uridine/uracil auxotrophic mutants in the filamentous fungus Aspergillus oryzae using Agrobacterium tumefaciens-mediated transformation

Cited by 34 publications

References 49 publications

Optimization of Production Conditions for Protoplasts and Polyethylene Glycol-Mediated Transformation of Gaeumannomyces tritici

Optimization of Production Conditions for Protoplasts and Polyethylene Glycol-Mediated Transformation of Gaeumannomyces tritici

Disruption or reduced expression of the orotidine-5′-decarboxylase gene pyrG increases citric acid production: a new discovery during recyclable genome editing in Aspergillus niger

Functional Genomics of Aspergillus oryzae: Strategies and Progress

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