An endogenous-signal-guided growth-coupled selection platform for glycosyltransferase evolution in Escherichia coli K5

Directed evolution of polysaccharide glycosyltransferases remains constrained by the absence of scalable selection outputs that report pathway-relevant enzyme function in living cells. Here, we developed an endogenous-signal-guided growth-coupled selection platform for glycosyltransferase evolution in Escherichia coli K5, a bacterium that naturally produces heparosan. By comparing a heparosan-deficient kfiA -deletion chassis with the parental strain, we prioritized the promoter upstream of ECK5_RS12660 (designated Pgnt) as a production-state-associated regulatory element. CRP was nominated as a functional amplifier of Pgnt output using DNA pull-down, proteomic prioritization, and inducible transcription-factor screening. To convert this endogenous regulatory response into a practical selection interface, we constructed a clean K5AC chassis lacking kfiA and kfiC , and benchmarked Pgnt-only and CRP-assisted circuits against NaKfiA variants across a defined activity gradient. Pgnt alone showed weak and non-monotonic reporter behavior, whereas CRP assistance generated a dual fluorescence/growth output that supported tetracycline-coupled enrichment of the highest-ranking benchmark variant. Targeted PmHS2 library selection further recovered recurrent substitutions, and reconstruction-based biochemical validation confirmed five activity-enhancing variants. Whole-genome diversification revealed a boundary condition for this selection logic, as the recovered K5-WGE strain did not show increased final heparosan production under the tested conditions. Analysis of endogenous kfiA / kfiC variants further showed that growth-coupled output can reflect complex-associated glycosyltransferase states rather than isolated KfiA catalytic activity alone. Because CRP was supplied from the same pBAD-controlled module in all benchmark and library comparisons, fluorescence and tetracycline-growth outputs were interpreted as circuit-level enrichment signals within a fixed assay configuration; productive mutations were assigned only after reconstruction and orthogonal biochemical validation. The Pgnt-CRP module provides an endogenous-signal-guided enrichment interface for targeted glycosyltransferase libraries. Its fluorescence and growth outputs integrate promoter activity, fixed CRP amplification, and host physiology; reconstruction and orthogonal biochemical validation are required to assign productive enzyme improvement.