Medium-chain fatty acids (MCFAs， 6-10 carbons) are valuable precursors to many industrial biofuels and chemicals， recently engineered reversal of the β-oxidation (r-BOX) cycle has been proposed as a potential platform for efficient synthesis of MCFAs. Previous studies have made many exciting achievements on functionally characterizing four core enzymes of this r-BOX cycle. However， the information about bottleneck nodes in this cycle is elusive. Here， a quantitative assessment of the inherent limitations of this cycle was conducted to capitalize on its potential. The selection of the core β-oxidation reversal enzymes in conjunction with acetyl-CoA synthetase endowed the ability to synthesize about 1g/L MCFAs. Furthermore， a gene dosage experiment was developed to identify two rate-limiting enzymes (acetyl-CoA synthetase and thiolase). The de novo pathway was then separated into two modules at thiolase and MCFA production titer increased to 2.8g/L after evaluating different construct environments. Additionally， the metabolism of host organism was reprogrammed to the desired biochemical product by the clustered regularly interspaced short palindromic repeats interference system， resulted in a final MCFA production of 3.8g/L. These findings described here identified the inherent limitations of r-BOX cycle and further unleashed the lipogenic potential of this cycle， thus paving the way for the development of a bacterial platform for microbial production of high-value oleo-chemicals from low-value carbons in a sustainable and environmentally friendly manner.