Turning a proof-of-concept synthetic biology design into a robust, high performing cell factory is a major time and money consuming task, which severely limits the growth of the white biotechnology sector. Here, we extend the use of tunable antibiotic resistance markers for synthetic evolution (TARSyn), a workflow for screening translation initiation region (TIR) libraries with antibiotic selection, to generic pathway engineering, and transform a proof-of-concept synbio design into a process that performs at industrially relevant levels. Using a combination of rational design and adaptive evolution, we recently engineered a high-performing bacterial strain for production of the important building block biochemical l-serine, based on two high-copy pET vectors facilitating expression of the serine biosynthetic genes serA, serC, and serB from three independent transcriptional units. Here, we prepare the bacterial strain for industrial scale up by transferring and reconfiguring the three genes into an operon encoded on a single low-copy plasmid. Not surprisingly, this initially reduces production titers considerably. We use TARSyn to screen both experimental and computational optimization designs resulting in high-performing synthetic serine operons and reach industrially relevant production levels of 50 g/L in fed-batch fermentations, the highest reported so far for serine production.