Abstract

Genome-scale and multigene transcriptional regulation are crucial technologies in metabolic engineering. However, in Escherichia coli , a stable and universal tool for whole-genome transcriptional activation, and an in situ tool for multigene regulation remain lacking. Here, we present CAGER, a versatile clustered regularly interspaced short palindromic repeats-associated transposases (CAST)-mediated gene regulation toolkit. Through rational mutagenesis, we mitigate the intrinsic transcriptional interference in the left end of CAST system derived from Vibrio cholerae . Using promoters or terminators as cargoes, CAGER constructs the genome-wide activation (3272 genes) or termination (3339 genes) libraries, from which new activation or inhibition targets relevant to cellular acetic acid assimilation are identified. Furthermore, with the aid of M13 phage and the promoter library, CAGER facilitates rapid in situ multigene expression diversification. Applied to lycopene synthesis, a library targeting seven genomic sites is constructed within 24 h, achieving a 73.6-fold yield increase. This work highlights the modifiability of CAST elements and broadens CAST’s application in transcriptional regulation.