Co-Directional Chromosomal Clustering of Metabolic Pathway Genes as a Layout Prior for Synthetic Construct Design.

Metabolic pathway design tools largely address reaction routes and enzyme choice, whereas a complementary set of questions-how pathway genes are arranged on bacterial chromosomes and whether co-directional neighborhoods can inform multi-gene construct layout-has received comparatively little systematic treatment. Here, we present SynPAL (Synteny-Informed Pathway Assembly and Layout), a computational analysis that scores co-directional clustering of BioCyc pathway genes from genomic coordinates (same replicon and strand, intergenic distance ≤ 2000 bp) and translates the resulting scores into design-oriented hypotheses. On a core panel of 55 prokaryotes (887 analyzable pathways), medium or high clustering occurs for 290 pathways (32.7%), with a mean cluster score of 0.432 versus 0.262 under an organism-matched random-gene-set null; 692/887 pathways remain significant after Benjamini-Hochberg false-discovery-rate control at q<0.05. High scores recover classical operons, including nan, bkd, pdxST, and gmd-fcl, under a fixed labeling protocol. Scoring gene-name order in pathway tables instead of coordinates yields substantially higher medium/high rates on the same pathways (80.1% vs. 32.0% on the full 55-organism panel; 90.5% vs. 49.2% on a paired 63-pathway set) and correlates only weakly with coordinate scores (Pearson's r=0.23), demonstrating that table order is not chromosomal synteny. Pathways are assigned design-readiness tiers T1-T4 as layout hypotheses, and sequence-backed construct drafts were generated for 222 of 224 T1 pathways. An expanded survey of 9071 prokaryotic databases (1629 pathways) shows a similar selective landscape (42.7% medium/high). SynPAL does not select enzymes, predict flux, or report wet-lab expression; it supplies coordinate-based cluster scores, tiers, and draft layouts that can accept a gene list from reaction-network CAD tools.