{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T11:18:53Z","timestamp":1788434333462,"version":"build-2803163510"},"reference-count":31,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T00:00:00Z","timestamp":1788393600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["BioTech"],"abstract":"<jats:p>Metabolic pathway design tools largely address reaction routes and enzyme choice, whereas a complementary set of questions\u2014how pathway genes are arranged on bacterial chromosomes and whether co-directional neighborhoods can inform multi-gene construct layout\u2014has 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 \u2264 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\u2013Hochberg false-discovery-rate control at q&lt;0.05. High scores recover classical operons, including nan, bkd, pdxST, and gmd\u2013fcl, 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\u2019s r=0.23), demonstrating that table order is not chromosomal synteny. Pathways are assigned design-readiness tiers T1\u2013T4 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.<\/jats:p>","DOI":"10.3390\/biotech15040075","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T10:10:33Z","timestamp":1788430233000},"page":"75","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Co-Directional Chromosomal Clustering of Metabolic Pathway Genes as a Layout Prior for Synthetic Construct Design"],"prefix":"10.3390","volume":"15","author":[{"given":"Xiaowei","family":"Han","sequence":"first","affiliation":[{"name":"College of Life Sciences, Northwest A&F University, Yangling 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"}]},{"given":"Zhixu","family":"Qiu","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Northwest A&F University, Yangling 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-2668-6336","authenticated-orcid":false,"given":"Jiani","family":"Hu","sequence":"additional","affiliation":[{"name":"School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"project-administration","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5595-1837","authenticated-orcid":false,"given":"Jie","family":"Song","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Northwest A&F University, Yangling 712100, China"},{"name":"School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"565","DOI":"10.1021\/sb4001273","article-title":"RetroPath: Automated Pipeline for Embedded Metabolic Circuits","volume":"3","author":"Carbonell","year":"2014","journal-title":"ACS Synth. Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1016\/j.ymben.2017.12.002","article-title":"RetroPath2.0: A retrosynthesis workflow for metabolic engineers","volume":"45","author":"Duigou","year":"2018","journal-title":"Metab. Eng."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1021\/acssynbio.9b00447","article-title":"Reinforcement Learning for Bioretrosynthesis","volume":"9","author":"Koch","year":"2020","journal-title":"ACS Synth. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Whitmore, L.S., Nguyen, B., Pinar, A., George, A., and Hudson, C.M. (2019). RetSynth: Determining all optimal and sub-optimal synthetic pathways that facilitate synthesis of target compounds in chassis organisms. BMC Bioinform., 20.","DOI":"10.1186\/s12859-019-3025-9"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"5082","DOI":"10.1038\/s41467-022-32661-x","article-title":"The automated Galaxy-SynBioCAD pipeline for synthetic biology design and engineering","volume":"13","author":"Duigou","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"318","DOI":"10.1016\/S0022-2836(61)80072-7","article-title":"Genetic regulatory mechanisms in the synthesis of proteins","volume":"3","author":"Jacob","year":"1961","journal-title":"J. Mol. Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1146\/annurev-biochem-013118-111914","article-title":"Designing Biological Circuits: Synthetic Biology Within the Operon Model and Beyond","volume":"90","author":"English","year":"2021","journal-title":"Annu. Rev. Biochem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2896","DOI":"10.1073\/pnas.96.6.2896","article-title":"The use of gene clusters to infer functional coupling","volume":"96","author":"Overbeek","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"324","DOI":"10.1016\/S0968-0004(98)01274-2","article-title":"Conservation of gene order: A fingerprint of proteins that physically interact","volume":"23","author":"Dandekar","year":"1998","journal-title":"Trends Biochem. Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"519","DOI":"10.1016\/j.mib.2004.08.006","article-title":"Order and disorder in bacterial genomes","volume":"7","author":"Rocha","year":"2004","journal-title":"Curr. Opin. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"6652","DOI":"10.1073\/pnas.110147297","article-title":"Operons in Escherichia coli: Genomic analyses and predictions","volume":"97","author":"Salgado","year":"2000","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"S329","DOI":"10.1093\/bioinformatics\/18.suppl_1.S329","article-title":"A powerful non-homology method for the prediction of operons in prokaryotes","volume":"18","year":"2002","journal-title":"Bioinformatics"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"679","DOI":"10.1016\/j.celrep.2015.12.085","article-title":"Operon gene order is optimized for ordered protein complex assembly","volume":"14","author":"Wells","year":"2016","journal-title":"Cell Rep."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1027","DOI":"10.1038\/nbt1226","article-title":"Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes","volume":"24","author":"Pfleger","year":"2006","journal-title":"Nat. Biotechnol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1241","DOI":"10.1038\/nbt.3063","article-title":"Functional optimization of gene clusters by combinatorial design and assembly","volume":"32","author":"Smanski","year":"2014","journal-title":"Nat. Biotechnol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"e98","DOI":"10.1093\/nar\/gkt151","article-title":"Spanning high-dimensional expression space using ribosome-binding site combinatorics","volume":"41","author":"Zelcbuch","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1843","DOI":"10.1093\/genetics\/143.4.1843","article-title":"Selfish operons: Horizontal transfer may drive the evolution of gene clusters","volume":"143","author":"Lawrence","year":"1996","journal-title":"Genetics"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"809","DOI":"10.1101\/gr.3368805","article-title":"Operon formation is driven by co-regulation and not by horizontal gene transfer","volume":"15","author":"Price","year":"2005","journal-title":"Genome Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1016\/j.ymben.2024.12.011","article-title":"Synthetic translational coupling system for accurate and predictable polycistronic gene expression control in bacteria","volume":"88","author":"Han","year":"2025","journal-title":"Metab. Eng."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1085","DOI":"10.1093\/bib\/bbx085","article-title":"The BioCyc collection of microbial genomes and metabolic pathways","volume":"20","author":"Karp","year":"2019","journal-title":"Brief. Bioinform."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"D445","DOI":"10.1093\/nar\/gkz862","article-title":"The MetaCyc database of metabolic pathways and enzymes\u2014A 2019 update","volume":"48","author":"Caspi","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Paley, S., and Karp, P.D. (2021). The BioCyc Metabolic Network Explorer. BMC Bioinform., 22.","DOI":"10.1186\/s12859-021-04132-5"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Midford, P.E., Cadigan, J., and Karp, P.D. (bioRxiv, 2024). Improved BioCyc Operon Prediction: Revisiting the Operon Prediction Problem, bioRxiv, preprint.","DOI":"10.1101\/2024.06.23.600222"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1038\/nmeth.1318","article-title":"Enzymatic assembly of DNA molecules up to several hundred kilobases","volume":"6","author":"Gibson","year":"2009","journal-title":"Nat. Methods"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e115","DOI":"10.1093\/nar\/gks596","article-title":"Primer3\u2014New capabilities and interfaces","volume":"40","author":"Untergasser","year":"2012","journal-title":"Nucleic Acids Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4806","DOI":"10.1128\/JB.185.16.4806-4815.2003","article-title":"Regulation of sialic acid catabolism by the DNA binding protein NanR in Escherichia coli","volume":"185","author":"Kalivoda","year":"2003","journal-title":"J. Bacteriol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2059","DOI":"10.1128\/JB.181.7.2059-2066.1999","article-title":"Role of BkdR, a transcriptional activator of the SigL-dependent isoleucine and valine degradation pathway in Bacillus subtilis","volume":"181","author":"Gardan","year":"1999","journal-title":"J. Bacteriol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1191","DOI":"10.1128\/JB.186.4.1191-1196.2004","article-title":"Physical and enzymological interaction of Bacillus subtilis proteins required for de novo pyridoxal 5\u2032-phosphate biosynthesis","volume":"186","author":"Belitsky","year":"2004","journal-title":"J. Bacteriol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"998","DOI":"10.1128\/JB.180.4.998-1001.1998","article-title":"Identification of the fucose synthetase gene in the colanic acid gene cluster of Escherichia coli K-12","volume":"180","author":"Andrianopoulos","year":"1998","journal-title":"J. Bacteriol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"5825","DOI":"10.1093\/molbev\/msab293","article-title":"eggNOG-mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale","volume":"38","author":"Cantalapiedra","year":"2021","journal-title":"Mol. Biol. Evol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"e02078-23","DOI":"10.1128\/mbio.02078-23","article-title":"Stability and gene strand bias of lambda prophages and chromosome organization in Escherichia coli","volume":"15","author":"Li","year":"2024","journal-title":"mBio"}],"container-title":["BioTech"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2673-6284\/15\/4\/75\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T10:46:48Z","timestamp":1788432408000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2673-6284\/15\/4\/75"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,3]]},"references-count":31,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2026,10]]}},"alternative-id":["biotech15040075"],"URL":"https:\/\/doi.org\/10.3390\/biotech15040075","relation":{},"ISSN":["2673-6284"],"issn-type":[{"value":"2673-6284","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,3]]}}}