Ouedraogo Jean Paul, Arentshorst Mark, Nikolaev Igor, Barends Sharief, Ram Arthur F J
Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Kluyver Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE Leiden, The Netherlands.
Dupont Industrial Biosciences, Archimedesweg 30, 2333 CN Leiden, The Netherlands.
J Biotechnol. 2016 Mar 20;222:25-8. doi: 10.1016/j.jbiotec.2016.02.012. Epub 2016 Feb 6.
We previously showed that creation of a double strand DNA break (DSB) by expressing I-SceI in an engineered Trichoderma reesei (Hypocrea jecorina) strain containing a I-SceI recognition site improved transformation and homologous integration efficiencies. In this study, we further improved homologous integration frequencies by combining I-SceI mediated double strand break with disruption of the tku70 gene. The inability of the tku70 mutant to repair a I-SceI mediated DSB via NHEJ was used to force integration of an expression cassette with homologous flanks surrounding the DSB site. Besides expressing I-SceI from a plasmid, we also show that adding I-SceI enzyme during transformation was successful to generate DSBs. The I-SceI enzyme mediated integration, or SEMI, in combination with a Δtku70 mutant has a synergistic effect on homologous recombination efficiencies as 90-100% of the transformants exhibited integration of the expression cassette at the homologous site.
我们之前表明，在含有I-SceI识别位点的工程化里氏木霉（枝状曲霉）菌株中表达I-SceI以产生双链DNA断裂（DSB），可提高转化和同源整合效率。在本研究中，我们通过将I-SceI介导的双链断裂与tku70基因的破坏相结合，进一步提高了同源整合频率。tku70突变体无法通过非同源末端连接（NHEJ）修复I-SceI介导的DSB，这被用于促使带有围绕DSB位点的同源侧翼的表达盒进行整合。除了从质粒表达I-SceI外，我们还表明在转化过程中添加I-SceI酶成功地产生了DSB。I-SceI酶介导的整合（SEMI）与Δtku70突变体相结合，对同源重组效率具有协同作用，因为90%-100%的转化体在同源位点表现出表达盒的整合。
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