posted on 2022-02-01, 18:24authored byShuai Ma, Tianyuan Su, Jinming Liu, Xuemei Lu, Qingsheng Qi
Genome
reduction is an important strategy in synthetic biology
for constructing functional chassis cells or minimal genomes. However,
the limited knowledge of complex gene functions and interactions makes
genome reduction by rational design encounter a bottleneck. Here,
we present an iterative and random genome reduction method for Escherichia coli, named “transposon-mediated
random deletion (TMRD)”. TMRD generates random double-strand
breaks (DSBs) in the genome by combining Tn5 transposition
with the CRISPR/Cas9 system and allows genomic deletions of various
sizes at random positions during DSB repair through the intracellular
alternative end-joining mechanism. Using E. coli MG1655 as the original strain, a pool of cells with multiple random
genomic deletions were obtained after five reduction cycles. The growth
rates of the obtained cells were comparable to that of MG1655, while
the electroporation efficiency increased by at least 2 magnitudes.
TMRD can generate a small E. coli library
carrying multiple and random genomic deletions while enriching the
cells with environmental fitness in the population. TMRD has the potential
to be widely applied in the construction of minimal genomes or chassis
cells for metabolic engineering.