Abstract
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.
Highlights
Dehydrogenase), adhE, brnQ and citE[8] or fumC and sucC[1] enhanced the malonyl-CoA concentration
To implement the clustered regularly interspaced short palindromic repeats interference (CRISPRi) platform in E. coli, a catalytically dead Cas[9] mutant derived from Streptococcus pyogenes cas[9] gene[15], which acts as an RNA-guided DNA-binding complex, was expressed under T7 promoter
Acetyl-CoA serves as the first flux control point for flavonoid biosynthesis, whereas malonyl-CoA serves as a starting point for the synthesis of flavonoids, which are only consumed for synthesizing fatty acids[5]
Summary
Dehydrogenase), adhE (acetaldehyde dehydrogenase), brnQ (branched chain amino acid transporter) and citE (citrate lyase)[8] or fumC (fumarate hydratase) and sucC (succinyl-CoA synthetase)[1] enhanced the malonyl-CoA concentration. We explored the impact of fine-tuning the central metabolic pathways by using a CRISPRi system[15] to enhance heterologous pathway productivity, using the production of (2S)-naringenin as a model system. Genes involved in central metabolic pathways were repressed by the CRISPRi system[15] to identify individual target genes that could increase the intracellular malonyl-CoA level by over 223%. The efficiency of repression of these target genes was tuned to balance malonyl-CoA generation and biomass accumulation (the final OD600 decreased by less than 10%). Multiple gene repressing was performed to achieve a high yield of (2S)-naringenin (421.6 mg/L) This strategy enhances the malonyl-CoA concentration without the need to add substrates for malonyl-CoA generation, which potentially provides an economically sustainable process for the efficient production of other plant natural compounds
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