Fine modulation of carbon flow in central carbon metabolism via ribosome-binding site modification in Escherichia coli

Optimization of flux distribution in central carbon metabolism is important to improve the microbial productivity. As the number of precursors required for synthesis differs for each target compound, optimal flux distribution also varies. A library of mutant strains with diverse flux distributions can aid in optimal strain screening. Therefore, in this study, we aimed to construct a library of <i>Escherichia coli</i> strains with stepwise changes in flux distribution by introducing mutations into the ribosome-binding sites of key enzyme genes on its chromosome. We focused on the flux ratios at the glucose-6-phosphate and acetyl-CoA branch points to enhance mevalonate production. Mutations were introduced into the ribosome-binding sites of <i>pgi</i> and <i>gltA</i> to vary the flux ratios of the two pathway branches. Furthermore, a combinatorial repression library comprising 16 strains was constructed by varying <i>pgi</i> and <i>gltA</i> expression at four levels, and a plasmid containing mevalonate synthesis genes was introduced into each strain. Batch cultures were performed to obtain strains with mevalonate titers and yields 2.4- and 3.4-fold higher than those of the parent strain. Overall, our combinatorial suppression library of <i>pgi</i> and <i>gltA</i> facilitated the effective identification of mutants with optimal metabolism for mevalonate production.