Construction of an Engineering Strain Producing High Yields of α-TransglucosidaseviaAgrobacterium tumefaciens-Mediated Transformation ofAsperillus niger

Li, Ming; Zhou, Ling; Li, Meng; Huang, Yue; Sun, Xin; Lu, Fuping

doi:10.1271/bbb.130281

Cited by 13 publications

(4 citation statements)

References 40 publications

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“…In conclusion, with the optimized approach, a high transformation efficiency of 145 transformants per 10 5 conidia was obtained. This was higher compared with the amount of transformants obtained in fungi using other approaches, typically less than 100 transformants per 10 5 conidia [ 18 , 29 – 31 ].…”

Section: Resultsmentioning

confidence: 93%

Development of genetic tools for Myceliophthora thermophila

Lin

et al. 2015

BMC Biotechnol

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BackgroundThe thermophilic filamentous fungus Myceliophthora thermophila has many suitable characteristics for industrial biotechnology and could be a promising new chassis system for synthetic biology, particularly the ATCC 42464 strain, whose genome was sequenced in 2011. However, metabolic engineering of this strain using genetic approaches has not been reported owing to a lack of genetic tools for this organism.ResultsIn the present study, we developed a high efficiency Agrobacterium tumefaciens mediated transformation system for M. thermophila, including an approach for targeted gene deletion using green fluorescence protein (GFP) as a marker for selection. Up to 145 transformants per 105 conidia were obtained in one transformation plate. Moreover, a ku70 deletion mutant was constructed in the ATCC 42464 background using the tools developed in present study and subsequently characterized. The ku70 deletion construct was designed using resistance to phosphinothricin as the selection marker. Additionally, a GFP-encoding cassette was incorporated that allowed for the selection of site-specific (no fluorescence) or ectopic (fluorescence) integration of the ku70 construct. Transformants with ectopically integrated ku70 deletion constructs were therefore identified using the fluorescent signal of GFP. PCR and Southern blotting analyses of non-fluorescent putative ku70 deletion transformants revealed all 11 tested transformants to be correct deletions. The deletion frequency in a pool of 116 transformants analyzed was 58 %. Moreover, the homologous rate improved about 3 folds under ku70 mutant using the pyrG as a test gene to disrupt in M. thermophila.ConclusionsWe successfully developed an efficient transformation and target gene disruption approach for M. thermophila ATCC 42464 mediated by A. tumefaciens. The tools and the ku70 deletion strain developed here should advance the development of M. thermophila as an industrial host through metabolic engineering and accelerate the elucidation of the mechanism of rapid cellulose degradation in this thermophilic fungus.

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Section: Resultsmentioning

confidence: 93%

Development of genetic tools for Myceliophthora thermophila

Lin

et al. 2015

BMC Biotechnol

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“…The level of gene expression strongly relied on the copy number of the gene in the host cell (Li et al, 2013). Our results showed that three selected strains harbored 2 or 3 copies of the phyA expression cassette in their genomes (Table 3).…”

Section: A Oryzae Aut1-pld Represents An Excellent Host For Heterologous Expressionmentioning

confidence: 79%

Heterologous phytase expression in the food filamentous fungus Aspergillus oryzae using the added rice husk cultivation model

Dung¹,

Tuấn²

2020

AJB

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Aspergillus oryzae, a safe filamentous fungus, is widely used in food and enzyme production. In this study, we examined a cultivation model using rice husks as carrier to assess the capacity of recombinant protein production in A. oryzae. The model was first tested with the A. oryzae strain expressing the DsRed reporter gene. Expression of DsRed was easily detected by the pink color of the fungal mycelium on culture media and under a fluorescence microscope. The model was then evaluated with the phyA gene encoding a phytase from the fungus Aspergillus fumigatus. The phyA expression cassette regulated by the amyB promoter was permanently integrated into the genome of A. oryzae via Agrobacterium tumefaciens-mediated transformation with the pyrG nutritional marker. Results showed that A. oryzae transgenic strains carrying 2−3 copies of the phyA gene in their genomes exhibited a significant increase in phytase activity on agar medium supplemented with phytate. With rice husks added, these transgenic strains could secrete the recombinant phytase into the culture and phytase activity of the crude enzyme solution increased by 4.3 times compared to the unstransgenic A. oryzae. The established cultivation model and the transgenic approach in this study represent a potential for being used in production of secreted recombinant enzymes for animal feeds.

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“…Three signal sequences were selected including the glucoamylase ( glaA ) signal sequence, the ANL signal sequence and the cellobiohydrolase I ( cbhI ) signal sequence for the expression of ANL in A. niger . The glaA signal sequence is used widely in A. niger , because glucoamylase is expressed in high amounts in A. niger [ 24 ], and the signal sequence from a highly expressed gene usually helps the extracellular expression of the target gene [ 25 ]. The ANL signal sequence is its own signal sequence.…”

Section: Resultsmentioning

confidence: 99%

Improved Homologous Expression of the Acidic Lipase from Aspergillus niger

Si-Yuan¹,

Xu²,

Xiang³

2020

Journal of Microbiology and Biotechnology

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In this study, the acidic lipase from Aspergillus niger (ANL) was homologously expressed in A. niger. The expression of ANL was significantly improved by the expression of the native ANL with the introns, the addition of the Kozak sequence and the optimization of the signal sequences. When the cDNA sequence of ANL fused with the glaA signal was expressed under the gpdA promoter in A. niger, no lipase activity could be detected. We then tried to improve the expression by using the full-length ANL gene containing three introns, and the lipase activity in the supernatant reached 75.80 U/ml, probably as a result of a more stable mRNA structure. The expression was further improved to 100.60 U/ml by introducing a Kozak sequence around the start codon due to a higher translation efficiency. Finally, the effects of three signal sequences including the cbhI signal, the ANL signal and the glaA signal on the lipase expression were evaluated. The transformant with the cbhI signal showed the highest lipase activity (314.67 U/ml), which was 1.90-fold and 3.13-fold higher than those with the ANL signal and the glaA signal, respectively. The acidic lipase was characterized and its highest activity was detected at pH 3.0 and a temperature of 45ºC. These results provided promising strategies for the production of the acidic lipase from A. niger.

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Construction of an Engineering Strain Producing High Yields of α-TransglucosidaseviaAgrobacterium tumefaciens-Mediated Transformation ofAsperillus niger

Cited by 13 publications

References 40 publications

Development of genetic tools for Myceliophthora thermophila

Development of genetic tools for Myceliophthora thermophila

Heterologous phytase expression in the food filamentous fungus <i> Aspergillus oryzae </i>using the added rice husk cultivation model

Improved Homologous Expression of the Acidic Lipase from Aspergillus niger

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