Abstract
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.