Escherichia coli BW25113 was metabolically engineered for CoQ(10) production by replacing ispB with ddsA from Gluconobacter suboxydans. Effects of precursor balance and reduced nicotinamide-adenine dinucleotide phosphate (NADPH) availability on CoQ(10) production in E. coli were investigated. The knockout of pykFA along with pck overexpression could maintain a balance between glyceraldehyde 3-phosphate and pyruvate, increasing CoQ(10) production. Replacement of native NAD-dependent gapA with NADP-dependent gapC from Clostridium acetobutylicum, together with the overexpression of gapC, could increase NADPH availability and then enhanced CoQ(10) production. Three effects, overexpressions of various genes in CoQ biosynthesis and central metabolism, different vectors and culture conditions on CoQ(10) production in E. coli, were all investigated. The investigation of different vectors indicated that low copy number vector may be more beneficial for CoQ(10) production in E. coli. The recombinant E. coli (Delta ispB::ddsA, Delta pykFA and Delta gapA::gapC), harboring the two plasmids encoding pck, dxs, idi and ubiCA genes under the control of P-T5 on pQE30, ispA, ddsA from Gluconobacter suboxydans and gapC from Clostridium acetobutylicum under the control of P-BAD on pBAD33, could produce CoQ(10) up to 3.24 mg.g(-1) dry cell mass simply by changing medium from M9YG to SOB with phosphate salt and initial culture pH from 7.0 to 5.5. The yield is unprecedented and 1.33 times of the highest production so far in E. coli.