产品介绍

 MUCICAT方法操作大肠杆菌，可以实现 5天内在基因组水平敲入10个拷贝的基因。基因组整合型重组菌株表达葡萄糖脱氢酶的

水平达到pET24a表达水平的2.6倍。而且MUCICAT方法可以应用于柠檬塔图姆氏菌，也预示了这个方法在其他细菌中

应用的潜力。

 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.

参考文献：

Multicopy Chromosomal Integration Using CRISPR-Associated Transposases.

ACS Synth. Biol. 2020, 9, 1998−2008

https://pubs.acs.org/doi/10.1021/acssynbio.0c00073

基本信息