Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China.
Henan Engineering Laboratory for Bioconversion Technology of Functional Microbes, College of Life Sciences, Henan Normal University, Xinxiang, 453007, Henan, China.
Microb Cell Fact. 2022 Sep 7;21(1):182. doi: 10.1186/s12934-022-01908-z.
The methylotrophic yeast Pichia pastoris is considered as an ideal host for the production of recombinant proteins and chemicals. However, low homologous recombination (HR) efficiency hinders its precise and extensive genetic manipulation. To enhance the homology-directed repair over non-homologous end joining (NHEJ), we expressed five exonucleases that were fused with the Cas9 for enhancing end resection of double strand breaks (DSBs) of DNA cuts.
The endogenous exonuclease Mre11 and Exo1 showed the highest positive rates in seamless deletion of FAA1, and fusing the MRE11 to the C-terminal of CAS9 had the highest positive rate and relatively high number of clones. We observed that expression of CAS9-MRE11 significantly improved positive rates when simultaneously seamless deletion of double genes (from 76.7 to 86.7%) and three genes (from 10.8 to 16.7%) when overexpressing RAD52. Furthermore, MRE11 overexpression significantly improved the genomic integration of multi-fragments with higher positive rate and clone number.
Fusion expression of the endogenous exonuclease Mre11 with Cas9 enhances homologous recombination efficiency in P. pastoris. The strategy described here should facilitate the metabolic engineering of P. pastoris toward high-level production of value-added compounds.
甲醇营养型酵母毕赤酵母被认为是生产重组蛋白和化学物质的理想宿主。然而，低同源重组（HR）效率阻碍了其精确和广泛的遗传操作。为了增强同源定向修复相对于非同源末端连接（NHEJ），我们表达了五个与 Cas9 融合的外切核酸酶，以增强 DNA 切口双链断裂（DSB）的末端切除。
内源性核酸外切酶 Mre11 和 Exo1 在 FAA1 的无缝缺失中表现出最高的阳性率，并且将 MRE11 融合到 CAS9 的 C 末端具有最高的阳性率和相对较高的克隆数。我们观察到当同时过表达 RAD52 时，CAS9-MRE11 的表达显著提高了双基因（从 76.7%提高到 86.7%）和三个基因（从 10.8%提高到 16.7%）的无缝缺失的阳性率。此外，MRE11 的过表达显著提高了多片段的基因组整合，具有更高的阳性率和克隆数。
Cas9 与内源性核酸外切酶 Mre11 的融合表达提高了毕赤酵母中的同源重组效率。这里描述的策略应该有助于毕赤酵母的代谢工程，以实现高附加值化合物的生产。
