| Title |
I-SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei
|
|---|---|
| Published in |
Applied Microbiology and Biotechnology, August 2015
|
| DOI | 10.1007/s00253-015-6829-1 |
| Pubmed ID | |
| Authors |
Jean Paul Ouedraogo, Mark Arentshorst, Igor Nikolaev, Sharief Barends, Arthur F. J. Ram |
| Abstract |
Targeted integration of expression cassettes for enzyme production in industrial microorganisms is desirable especially when enzyme variants are screened for improved enzymatic properties. However, currently used methods for targeted integration are inefficient and result in low transformation frequencies. In this study, we expressed the Saccharomyces cerevisiae I-SceI meganuclease to generate double-strand breaks at a defined locus in the Trichoderma reesei genome. We showed that the double-strand DNA breaks mediated by I-SceI can be efficiently repaired when an exogenous DNA cassette flanked by regions homologous to the I-SceI landing locus was added during transformation. Transformation efficiencies increased approximately sixfold compared to control transformation. Analysis of the transformants obtained via I-SceI-mediated gene targeting showed that about two thirds of the transformants resulted from a homologous recombination event at the predetermined locus. Counter selection of the transformants for the loss of the pyrG marker upon integration of the DNA cassette showed that almost all of the clones contained the cassette at the predetermined locus. Analysis of independently obtained transformants using targeted integration of a glucoamylase expression cassette demonstrated that glucoamylase production among the transformants was high and showing limited variation. In conclusion, the gene targeting system developed in this study significantly increases transformation efficiency as well as homologous recombination efficiency and omits the use of Δku70 strains. It is also suitable for high-throughput screening of enzyme variants or gene libraries in T. reesei. |
X Demographics
The data shown below were collected from the profile of 1 X user who shared this research output. Click here to find out more about how the information was compiled.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| France | 1 | 100% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Members of the public | 1 | 100% |
Mendeley readers
The data shown below were compiled from readership statistics for 56 Mendeley readers of this research output. Click here to see the associated Mendeley record.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Denmark | 1 | 2% |
| Unknown | 55 | 98% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Researcher | 14 | 25% |
| Student > Master | 7 | 13% |
| Student > Ph. D. Student | 5 | 9% |
| Student > Bachelor | 4 | 7% |
| Student > Doctoral Student | 3 | 5% |
| Other | 7 | 13% |
| Unknown | 16 | 29% |
| Readers by discipline | Count | As % |
|---|---|---|
| Agricultural and Biological Sciences | 16 | 29% |
| Biochemistry, Genetics and Molecular Biology | 11 | 20% |
| Engineering | 3 | 5% |
| Medicine and Dentistry | 2 | 4% |
| Environmental Science | 1 | 2% |
| Other | 4 | 7% |
| Unknown | 19 | 34% |
Attention Score in Context
This research output has an Altmetric Attention Score of 4. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 23 February 2023.
All research outputs
#6,908,521
of 24,119,703 outputs
Outputs from Applied Microbiology and Biotechnology
#2,404
of 8,034 outputs
Outputs of similar age
#76,885
of 267,525 outputs
Outputs of similar age from Applied Microbiology and Biotechnology
#29
of 161 outputs
Altmetric has tracked 24,119,703 research outputs across all sources so far. This one has received more attention than most of these and is in the 70th percentile.
So far Altmetric has tracked 8,034 research outputs from this source. They receive a mean Attention Score of 4.3. This one has gotten more attention than average, scoring higher than 69% of its peers.
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We're also able to compare this research output to 161 others from the same source and published within six weeks on either side of this one. This one has done well, scoring higher than 80% of its contemporaries.