A Dual Selection Marker Transformation System Using Agrobacterium tumefaciens for the Industrial Aspergillus oryzae 3.042

Sun, Yunlong; Niu, Yinong; He, Bin; Ma, Ling; Li, Ganghua; Tran, Van‐Tuan; Hú, Zhìhóng

doi:10.4014/jmb.1811.11027

Cited by 17 publications

(12 citation statements)

References 4 publications

(6 reference statements)

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“…A clear insight into fatty acid metabolism will contribute to a better understanding of ethanol tolerance mechanisms and facilitate the construction of industrial strains with a higher ethanol tolerance. Recent advances in the genetic manipulation of A. oryzae using Agrobacterium tumefaciens -mediated transformation (ATMT) could facilitate this goal [42,43].…”

Section: Discussionmentioning

confidence: 99%

Modulation of Fatty Acid Composition of Aspergillus oryzae in Response to Ethanol Stress

Hú

et al. 2019

Microorganisms

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The koji mold Aspergillus oryzae is widely adopted for producing rice wine, wherein koji mold saccharifies rice starch and sake yeast ferments glucose to ethanol. During rice wine brewing, the accumulating ethanol becomes a major source of stress for A. oryzae, and there is a decline in hydrolysis efficiency. However, the protective mechanisms of A. oryzae against ethanol stress are poorly understood. In the present study, we demonstrate that ethanol adversity caused a significant inhibition of mycelium growth and conidia formation in A. oryzae, and this suppressive effect increased with ethanol concentration. Transmission electron microscopy analysis revealed that ethanol uptake triggered internal cellular perturbations, such as irregular nuclei and the aggregation of scattered vacuoles in A. oryzae cells. Metabolic analysis uncovered an increase in fatty acid unsaturation under high ethanol conditions, in which a large proportion of stearic acid was converted into linoleic acid, and the expression of related fatty acid desaturases was activated. Our results therefore improve the understanding of ethanol adaptation mechanisms in A. oryzae and offer target genes for ethanol tolerance enhancement via genetic engineering.

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Section: Discussionmentioning

confidence: 99%

Modulation of Fatty Acid Composition of Aspergillus oryzae in Response to Ethanol Stress

Hú

et al. 2019

Microorganisms

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“…Aspergillus oryzae 3.042 (CICC 40092) was obtained from the China Center of Industry Culture Collection (Beijing, China) and used as the wild type strain. The A. oryzae 3.042 aaapyrG was constructed using Agrobacterium tumefaciens -mediated transformation in our laboratory (Sun et al, 2019). A. oryzae was cultured on (Czapek-Dox Agar) medium (2% sucrose, 0.2% NaNO 3 , 0.1% KH 2 PO 4 , 0.05% MgSO 4 , 0.05% KCl, 0.05% NaCl, 0.002% FeSO 4 , 1.6% agar, pH 5.5) at 30°C.…”

Section: Methodsmentioning

confidence: 99%

“…All the constructed vectors were transformed into A. tumefaciens AGL1. Then, the vectors were transformed into A. oryzae 3.042 aaapyrG according to the procedure published by our laboratory (Sun et al, 2019). Single spores of the transformants were cultured for further analysis.…”

Section: Methodsmentioning

confidence: 99%

“…The peroxisome targeted signal (PTS1), SRL amino acid, was fused to the C-terminal of GFP using PCR to visualize peroxisomes (Escano et al, 2009). Then, the GFP sequence of pEX1- ptr A (Nguyen et al, 2016) was replaced with GFP-PTS1 to construct pEX1- ptr A-GFP-PTS1 (Sun et al, 2019). The first 72 amino acids of AoCit1 protein was used as the mitochondria targeted signal (MTS) (Takaya et al, 2009).…”

Section: Methodsmentioning

confidence: 99%

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Mevalonate Diphosphate Decarboxylase MVD/Erg19 Is Required for Ergosterol Biosynthesis, Growth, Sporulation and Stress Tolerance in Aspergillus oryzae

et al. 2019

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Mevalonate diphosphate decarboxylase (MVD; EC 4.1.1.33) is a key enzyme of the mevalonic acid (MVA) pathway. In fungi, the MVA pathway functions as upstream of ergosterol biosynthesis, and MVD is also known as Erg19. Previously, we have identified Aoerg19 in Aspergillus oryzae using bioinformatic analysis. In this study, we showed that AoErg19 function is conserved using phylogenetic analysis and yeast complementation assay. Quantitative reverse transcription–PCR (qRT-PCR) indicated that Aoerg19 expression changed in different growth stages and under different forms of abiotic stress. Subcellular localization analysis showed that AoErg19 was located in the vacuole. Overexpression of Aoerg19 decreased the ergosterol content in A. oryzae, which may due to the feedback-mediated downregulation of Aoerg8 . Consistent with the decrease in ergosterol content, both Aoerg19 overexpression and RNAi strains of A. oryzae are sensitive to abiotic stressors, including ergosterol biosynthesis inhibitor, temperature, salt and ethanol. Thus, we have identified the function of AoErg19 in A. oryzae , which may assist in genetic modification of MVA and the ergosterol biosynthesis pathway.

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“…In 2017, Nguyen et al further confirmed that the use of AMT with the pyrG selectable marker is a powerful and efficient method for genetic transformation and recombinant gene expression studies in A. oryzae [20]. According to the AMT method introduced by Nguyen et al, our group established a dual selection marker transformation system using AMT for A. oryzae 3042 [23]. The AMT method is more efficient and simpler to implement due to the direct use of fungal spores as the material, avoiding the need to obtain protoplasts, which is laborious and requires a tricky procedure.…”

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 Dual Selection Marker Transformation System Using Agrobacterium tumefaciens for the Industrial Aspergillus oryzae 3.042

Cited by 17 publications

References 4 publications

Modulation of Fatty Acid Composition of Aspergillus oryzae in Response to Ethanol Stress

Modulation of Fatty Acid Composition of Aspergillus oryzae in Response to Ethanol Stress

Mevalonate Diphosphate Decarboxylase MVD/Erg19 Is Required for Ergosterol Biosynthesis, Growth, Sporulation and Stress Tolerance in Aspergillus oryzae

Functional Genomics of Aspergillus oryzae: Strategies and Progress

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