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.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
Junjun
J
Wu
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
Guocheng
G
Du
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
Jian
J
Chen
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
Jingwen
J
Zhou
D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D044950:Flavanones / Q000737:chemistry / Q000378:metabolism; D005419:Flavonoids / Q000096:biosynthesis*; D008316:Malonyl Coenzyme A / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism
D029968:Escherichia coli Proteins; D044950:Flavanones; D005419:Flavonoids; D008316:Malonyl Coenzyme A; C005273:naringenin