Multicopy Chromosomal Integration Using CRISPR-Associated Transposases.,ACS Synthetic Biology

当前位置： X-MOL 学术 › ACS Synth. Biol. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Multicopy Chromosomal Integration Using CRISPR-Associated Transposases.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-06-17 , DOI: 10.1021/acssynbio.0c00073
Yiwen Zhang _{1,

2} , Xiaoman Sun ₃ , Qingzhuo Wang _{1,

2} , Jiaqi Xu ₄ , Feng Dong ₅ , Siqi Yang _{1,

2} , Jiawei Yang ₃ , Zixu Zhang ₃ , Yuan Qian ₁ , Jun Chen ₁ , Jiao Zhang _{1,

2} , Yingmiao Liu ₁ , Rongsheng Tao ₅ , Yu Jiang ₆ , Junjie Yang ₁ , Sheng Yang _{1,

5}

Affiliation

Controlling the copy number of gene expression cassettes is an important strategy to engineer bacterial cells into high-efficiency biocatalysts. Current strategies mostly use plasmid vectors, but multicopy plasmids are often genetically unstable, and their copy numbers cannot be precisely controlled. The integration of expression cassettes into a bacterial chromosome has advantages, but iterative integration is laborious, and it is challenging to obtain a library with varied gene doses for phenotype characterization. Here, we demonstrated that multicopy chromosomal integration using CRISPR-associated transposases (MUCICAT) can be achieved by designing a crRNA to target multicopy loci or a crRNA array to target multiple loci in the Escherichia coli genome. Within 5 days without selection pressure, E. coli strains carrying cargos with successively increasing copy numbers (up to 10) were obtained. Recombinant MUCICAT E. coli containing genomic multicopy glucose dehydrogenase expression cassettes showed 2.6-fold increased expression of this important industrial enzyme compared to E. coli harboring the conventional protein-expressing plasmid pET24a. Successful extension of MUCICAT to Tatumella citrea further demonstrated that MUCICAT may be generally applied to many bacterial species.

中文翻译：

使用CRISPR相关转座酶进行多拷贝染色体整合。

控制基因表达盒的拷贝数是将细菌细胞改造成高效生物催化剂的重要策略。当前的策略大多使用质粒载体，但是多拷贝质粒通常在遗传上是不稳定的，并且其拷贝数不能精确控制。将表达盒整合到细菌染色体中具有优势，但是迭代整合是费力的，并且获得具有用于表型表征的不同基因剂量的文库是具有挑战性的。在这里，我们证明了使用CRISPR相关转座酶（MUCICAT）进行多拷贝染色体整合可以通过设计靶向多拷贝基因座的crRNA或靶向大肠杆菌基因组中多个基因座的crRNA阵列来实现。5天之内没有选择压力，获得携带货物的大肠杆菌菌株，其拷贝数连续增加（最多10个）。包含基因组多拷贝葡萄糖脱氢酶表达盒的重组MUCICAT大肠杆菌显示，与具有传统蛋白表达质粒pET24a的大肠杆菌相比，这种重要的工业酶的表达增加了2.6倍。MUCICAT成功地扩展到了柠檬土木霉菌进一步证明了MUCICAT通常可以应用于许多细菌。

更新日期：2020-08-21

点击分享查看原文

点击收藏

阅读更多本刊最新论文本刊介绍/投稿指南11