Identification of bottlenecks in Escherichia coli engineered for the production of CoQ(10) |
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Authors: | Cluis Corinne P Ekins Andrew Narcross Lauren Jiang Heng Gold Nicholas D Burja Adam M Martin Vincent J J |
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Institution: | aDepartment of Biology, Concordia University, 7141 Sherbrooke West, Montréal, Québec, Canada H4B 1R6;bOcean Nutrition Canada, Fermentation and Metabolic Engineering Group, 101 Research Drive, Dartmouth, Nova Scotia, Canada B2Y 4T6 |
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Abstract: | In this work, Escherichia coli was engineered to produce a medically valuable cofactor, coenzyme Q10 (CoQ10), by removing the endogenous octaprenyl diphosphate synthase gene and functionally replacing it with a decaprenyl diphosphate synthase gene from Sphingomonas baekryungensis. In addition, by over-expressing genes coding for rate-limiting enzymes of the aromatic pathway, biosynthesis of the CoQ10 precursor para-hydroxybenzoate (PHB) was increased. The production of isoprenoid precursors of CoQ10 was also improved by the heterologous expression of a synthetic mevalonate operon, which permits the conversion of exogenously supplied mevalonate to farnesyl diphosphate. The over-expression of these precursors in the CoQ10-producing E. coli strain resulted in an increase in CoQ10 content, as well as in the accumulation of an intermediate of the ubiquinone pathway, decaprenylphenol (10P-Ph). In addition, the over-expression of a PHB decaprenyl transferase (UbiA) encoded by a gene from Erythrobacter sp. NAP1 was introduced to direct the flux of DPP and PHB towards the ubiquinone pathway. This further increased CoQ10 content in engineered E. coli, but decreased the accumulation of 10P-Ph. Finally, we report that the combined over-production of isoprenoid precursors and over-expression of UbiA results in the decaprenylation of para-aminobenzoate, a biosynthetic precursor of folate, which is structurally similar to PHB. |
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Keywords: | Abbreviations: CoQ10 coenzyme Q10 DPP decaprenyl diphosphate DPS decaprenyl diphosphate synthase PABA para-aminobenzoate PHB para-hydroxybenzoate 10P-AB 3-decaprenyl-4-aminobenzoate 10P-HB 3-decaprenyl-4-hydroxybenzoate 10P-Ph decaprenylphenol SA shikimic acid |
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