Clustered Regularly Interspaced Short Palindromic Repeats Interference System Research Articles

Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli.

The limited supply of intracellular malonyl-CoA in Escherichia coli impedes the biological synthesis of polyketides, flavonoids and biofuels. Here, a clustered regularly interspaced short palindromic repeats (CRISPR) interference system was constructed for fine-tuning central metabolic pathways to efficiently channel carbon flux toward malonyl-CoA. Using synthetic sgRNA to silence candidate genes, genes that could increase the intracellular malonyl-CoA level by over 223% were used as target genes. The efficiencies of repression of these genes were tuned to achieve appropriate levels so that the intracellular malonyl-CoA level was enhanced without significantly altering final biomass accumulation (the final OD600 decreased by less than 10%). Based on the results, multiple gene repressing was successful in approaching the limit of the amount of malonyl-CoA needed to produce the plant-specific secondary metabolite (2S)-naringenin. By coupling the genetic modifications to cell growth, the combined effects of these genetic perturbations increased the final (2S)-naringenin titer to 421.6 mg/L, which was 7.4-fold higher than the control strain. The strategy described here could be used to characterize genes that are essential for cell growth and to develop E. coli as a well-organized cell factory for producing other important products that require malonyl-CoA as a precursor.