Development of a new Agrobacterium-mediated transformation system based on a dual auxotrophic approach in the filamentous fungus Aspergillus oryzae

Thai, Hanh-Dung; Nguyen, Bich-Phuong Thi; Nguyen, Viet Tuyen; Nguyen, Quoc Tuan; Tran, Van‐Tuan

doi:10.1007/s11274-021-03060-z

Cited by 9 publications

(5 citation statements)

References 38 publications

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“…(Fiedler et al, 2017). A dual auxotrophic Agrobacterium tumefaciens-mediated transformation method with two selection markers -hisB and pryGhas also been constructed in A. oryzae (Thai et al, 2021). Additionally, ScLYS2 invovled in lysine biosynthesis is commonly utilized in genetic studies as a nutritional selectable marker in S. cerevisiae (Eriksson et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

TrLys9 participates in fungal development and lysine biosynthesis in <i>Trichoderma reesei</i>

Lan

Zhang

Gao

et al. 2023

J. Gen. Appl. Microbiol.

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Fungi uniquely synthesize lysine through the α-aminoadipate pathway. The saccharopine reductase ScLys9 catalyzes the formation of saccharopine from ɑ-aminoadipate 6-semialdehyde, the seventh step in the lysine biosynthesis pathway in Saccharomyces cerevisiae. Here, we characterized the functions of TrLys9, an ortholog of S. cerevisiae ScLys9 in the industrial filamentous fungus Trichoderma reesei. Transcription-level analysis indicated that TrLYS9 expression was higer in the conidia stage than in other stages. Disruption of TrLYS9 led to lysine auxotrophy. Phenotype analysis of the ΔTrlys9 mutant showed that TrLYS9 was involved in fungal development including vegetative growth, conidiation, and conidial germination and lysine biosynthesis. Cellulase production was also impaired in the ΔTrlys9 mutant due to the failure of conidial germination in liquid cellulase-inducing liquid medium. Defects in radial growth and asexual development of the ΔTrlys9 mutant were fully recovered when exogenous lysine was added to the medium. These results imply that TrLys9 is involved in fungal development and lysine biosynthesis in T. reesei.

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Section: Discussionmentioning

confidence: 99%

TrLys9 participates in fungal development and lysine biosynthesis in <i>Trichoderma reesei</i>

Lan

Zhang

Gao

et al. 2023

J. Gen. Appl. Microbiol.

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“…All the strains used in this study are described in Table 1 . All the strains are maintained on PDA slants with/without 10 mmol/ml uridine and 0.75 mg/ml 5-FOA at 28°C (Thai et al, 2021 ).…”

Section: Methodsmentioning

confidence: 99%

Construction of gene modification system with highly efficient and markerless for Monascus ruber M7

Chen

2022

Front. Microbiol.

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Monascus spp. are traditional medicinal and edible filamentous fungi in China, and can produce various secondary metabolites, such as Monascus pigments (MPs) and citrinin (CIT). Genetic modification methods, such as gene knock-out, complementation, and overexpression, have been used extensively to investigate the function of related genes in Monascus spp.. However, the resistance selection genes that can have been used for genetic modification in Monascus spp. are limited, and the gene replacement frequency (GRF) is usually <5%. Therefore, we are committed to construct a highly efficient gene editing system without resistance selection marker gene. In this study, using M. ruber M7 as the starting strain, we successfully constructed a so-called markerlessly and highly genetic modification system including the mutants ΔmrpyrGΔmrlig4 and ΔmrpyrGΔmrlig4::mrpyrG, in which we used the endogenous gene mrpyrG from M. ruber M7 instead of the resistance marker gene as the screening marker, and simultaneously deleted mrlig4 related to non-homologous end joining in M. ruber M7. Then, the morphology, the growth rate, the production of MPs and CIT of the mutants were analyzed. And the results show that the mutant strains have normal mycelia, cleistothecia and conidia on PDA+Uridine(U) plate, the biomass of each mutant is also no different from M. ruber M7. However, the U addition also has a certain effect on the orange and red pigments yield of M. ruber M7, which needs our further study. Finally, we applied the system to delete multiple genes from M. ruber M7 separately or continuously without any resistance marker gene, and found that the average GRF of ΔmrpyrGΔmrlig4 was about 18 times of that of M. ruber M7. The markerlessly and highly genetic modification system constructed in current study not only will be used for multi-gene simultaneous modification in Monascus spp., and also lays a foundation for investigating the effects of multi-genes modification on Monascus spp..

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“…We have previously shown that the glyceraldehyde 3-phosphate dehydrogenase promoter P gpdA and α amylase promoter PamyB in A. oryzae could not drive the expression of GFP or DsRed reporters in C. militaris [ 27 ]. Thus, the promoter of the reporter gene in pEX2D was replaced with the IF promoter, the promoter of the extension factor, in C. militaris to construct the IF-pEX2D vector [ 26 , 27 ]. Then, IF-pEX2D was transformed into A. tumefaciens and C. militaris .…”

Section: Microbial Strains and Plasmidsmentioning

confidence: 99%

“…The histidine auxotrophic marker has also been used as a selection marker for screening filamentous fungi transformants [ 25 ]. The hisB gene, which is essential for histidine biosynthesis, is a reliable selection marker for genetic transformation in A. oryzae [ 26 ].…”

Section: Introductionmentioning

confidence: 99%

Construction of a New Agrobacterium tumefaciens-Mediated Transformation System based on a Dual Auxotrophic Approach in Cordyceps militaris

Yan,

Shang,

Wang

et al. 2024

J. Microbiol. Biotechnol.

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Cordyceps militaris is a significant edible fungus that produces a variety of bioactive compounds. We have previously established a uridine/uracil auxotrophic mutant and a corresponding Agrobacterium tumefaciens -mediated transformation (ATMT) system for genetic characterization in C. militaris using pyrG as a screening marker. In this study, we constructed an ATMT system based on a dual pyrG and hisB auxotrophic mutant of C. militaris . Using the uridine/uracil auxotrophic mutant as the background and pyrG as a selection marker, the hisB gene encoding imidazole glycerophosphate dehydratase, required for histidine biosynthesis, was knocked out by homologous recombination to construct a histidine auxotrophic C. militaris mutant. Then, pyrG in the histidine auxotrophic mutant was deleted to construct a Δ pyrG Δ hisB dual auxotrophic mutant. Further, we established an ATMT transformation system based on the dual auxotrophic C. militaris by using GFP and DsRed as reporter genes. Finally, to demonstrate the application of this dual transformation system for studies of gene function, knock out and complementation of the photoreceptor gene Cm WC-1 in the dual auxotrophic C. militaris were performed. The newly constructed ATMT system with histidine and uridine/uracil auxotrophic markers provides a promising tool for genetic modifications in the medicinal fungus C. militaris .

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Development of a new Agrobacterium-mediated transformation system based on a dual auxotrophic approach in the filamentous fungus Aspergillus oryzae

Cited by 9 publications

References 38 publications

TrLys9 participates in fungal development and lysine biosynthesis in <i>Trichoderma reesei</i>

TrLys9 participates in fungal development and lysine biosynthesis in <i>Trichoderma reesei</i>

Construction of gene modification system with highly efficient and markerless for Monascus ruber M7

Construction of a New Agrobacterium tumefaciens-Mediated Transformation System based on a Dual Auxotrophic Approach in Cordyceps militaris

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