Fine-tuning the expression level of an engineered pathway is crucial for the metabolic engineering of a host toward a desired phenotype. However, most engineered hosts suffer from nonfunctional protein expression, metabolic imbalance, cellular burden or toxicity from intermediates when an engineered pathway is first introduced, which can decrease production of the desired product. To circumvent these obstacles, we developed a self-regulation system utilizing the trc/tac promoter, LacI(q) protein and ribosomal binding sites (RBS). With the purpose of improving coenzyme Q(10) (CoQ(10)) production by increasing the decaprenyl diphosphate supplement, enzymes DXS, DXR, IDI, and IspD were constitutively overexpressed under the control of the trc promoter in Rhodobacter sphaeroides. Then, a self-regulation system combining a set of RBSs for adjusting the expression of the LacI(q) protein was applied to tune the expression of the four genes, resulting in improved CoQ(10) production. Finally, another copy of the tac promoter with the UbiG gene (involved in the ubiquinone pathway of CoQ(10) biosynthesis) was introduced into the engineered pathway. By optimizing the expression level of both the upstream and downstream pathway, CoQ(10) production in the mutants was improved up to 93.34mg/L (7.16mg/g DCW), about twofold of the wild-type (48.25mg/L, 3.24mg/g DCW). Biotechnol. Bioeng. 2014;111: 761-769. (c) 2013 Wiley Periodicals, Inc.