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Sci Rep. 2015 Sep 1;5:13477. doi: 10.1038/srep13477.

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

Wu J1,2, Du G1,2, Chen J1,2, Zhou J1,2.

Author information

1
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
2
Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.

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.

PMID:
26323217
PMCID:
PMC4555050
DOI:
10.1038/srep13477
[Indexed for MEDLINE]
Free PMC Article

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