Genome-Scale Engineering Importing Property of Escherichia coli for Improving Production of 3-Hydroxypropionic Acid.

Microbial biochemical production can suffer reduced growth and productivity from intracellular product accumulation, which can be mitigated by minimizing product import. Limited understanding of import-related genes, especially for non-native products, has hindered this approach in strain development. We developed a workflow to identify genes involved in 3-hydroxypropionic acid (3-HP) import. We constructed a genome-wide overexpression library coupled with a 3-HP-responsive fluorescent biosensor and used flow cytometry to isolate narQ-overexpressing strains with a 3.0-fold higher fluorescence signal. Transcriptome profiling under NarQ overexpression revealed a distinct set of membrane-associated genes (acrD, mliC, and pgaABCD) that were transcriptionally upregulated, and functional tests confirmed that their overexpression enhanced 3-HP import while their deletion in producing strains increased 3-HP titers by up to 21% compared with the control strain. This study provides a systematic workflow for identifying import-related genes directly from genomic DNA, advancing the development of more efficient microbial production platforms.