Abstract Background dTDP-rhamnose is synthesized by four genes ( rfbBDAC ) in Escherichia coli . While reconfiguring this operon could potentially enhance biosynthetic yields, precisely quantifying intracellular dTDP-rhamnose levels remains technically challenging. In this study, we employed plant glycosyltransferases ( AtUGT89C1 and AtUGT78D1 ) as sensitive metabolic reporters, utilizing the production of flavonoid rhamnosides as a proxy to accurately assess the metabolic flux of the rfb gene cluster. By systematically reorganizing the rfb operon through gene rearrangement and the introduction of intergenic promoters, we identified the optimal genetic configuration for maximized dTDP-rhamnose biosynthesis. Results E. coli was successfully engineered for high-titer production of rhamnosylated flavonoids using AtUGT89C1 and AtUGT78D1 as metabolic reporters. We demonstrated that the spatial arrangement and transcriptional control of the rfb genes are as critical as enzyme selection. Our optimized pA- rfbBD - rfbAC construct—featuring a dual-promoter system inspired by a natural 58-bp intergenic gap—significantly outperformed the native operon, yielding 81.9 mg/L of genistein 7- O -rhamnoside and 241.1 mg/L of quercetin 3- O -rhamnoside. Conclusions A key finding is that the rate-limiting step of the rfb pathway is context-dependent, shifting between rfbC and rfbB depending on the gene configuration. While unbalanced constructs (e.g., pA- rfbDCBA ) showed dramatic improvements upon individual gene supplementation, the optimized pA- rfbBD - rfbAC configuration reached a state of metabolic harmony where further overexpression yielded diminishing returns. Our results suggest that maintaining a “modular operon” structure is superior to complete fragmentation or reversal. This work provides a strategic template for optimizing complex nucleotide-sugar pathways by leveraging natural genomic cues to bypass metabolic bottlenecks.
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