Background The rapidly expanding synthetic biology toolbox allows engineers to develop smarter strategies to tackle the optimization of complex biosynthetic pathways. In such a strategy, multi-gene pathways are subdivided in several modules which are each dynamically controlled to fine-tune their expression in response to a changing cellular environment. To fine-tune separate modules without interference between modules or from the host regulatory machinery, a sigma factor (σ) toolbox was developed in previous work for tunable orthogonal gene expression. Here, this toolbox is implemented in E. coli to orthogonally express and fine-tune a pathway for the heterologous biosynthesis of the industrially relevant plant metabolite, naringenin. To optimize the production of this pathway, a practical workflow is still imperative to balance all steps of the pathway. This is tackled here by the biosensor-driven screening, subsequent genotyping of combinatorially engineered libraries and finally the training of three different computer models to predict the optimal pathway configuration. Results The efficiency and knowledge gained through this workflow is demonstrated here by improving the naringenin production titer by 32% with respect to a random pathway library screen. Our best strain was cultured in a batch bioreactor experiment and was able to produce 286 mg/L naringenin from glycerol in approximately 26 h. This is the highest reported naringenin production titer in E. coli without the supplementation of pathway precursors to the medium or any precursor pathway engineering. In addition, valuable pathway configuration preferences were identified in the statistical learning process, such as specific enzyme variant preferences and significant correlations between promoter strength at specific steps in the pathway and titer. Conclusions An efficient strategy, powered by orthogonal expression, was applied to successfully optimize a biosynthetic pathway for microbial production of flavonoids in E. coli up to high, competitive levels. Within this strategy, statistical learning techniques were combined with combinatorial pathway optimization techniques and an in vivo high-throughput screening method to efficiently determine the optimal operon configuration of the pathway. This “pathway architecture designer” workflow can be applied for the fast and efficient development of new microbial cell factories for different types of molecules of interest while also providing additional insights into the underlying pathway characteristics.