Abstract
<title>Abstract</title> <p>Spatial organization of metabolic enzymes into multi-enzyme complexes is a powerful strategy to enhance biosynthetic flux and product yield in microbial cell factories. However, the current repertoire of orthogonal protein–protein interaction modules available for synthetic enzyme assembly remains limited. Here, we explore SNARE-derived α-helical coiled-coil domains from Saccharomyces cerevisiae as a new class of programmable interaction modules for metabolic engineering. Multiple sequence alignment of yeast SNARE coiled-coil regions revealed strongly conserved heptad residues, and AlphaFold3 modelling predicted high interaction propensity (ipTM > 0.6) for selected t-SNARE/v-SNARE peptide pairs. The predicted interactions were validated in vivo in E. coli by confocal microscopy: fusion of Vam3, Vam7, or Vti1 t-SNARE peptides to eGFP and of the v-SNARE peptide Nyv1 to mCherry produced co-localized punctate foci, in stark contrast to the diffuse cytoplasmic distribution of the unfused fluorophores. We then applied this modular assembly strategy to the heterologous astaxanthin biosynthetic pathway by tethering the carotenoid β-hydroxylase CrtZ and the carotenoid ketolase CrtW through SNARE coiled-coil peptide pairs. The best-performing strain, Z-vam3-W-Nyv1, produced 1 mg/g DCW of astaxanthin, a 2-fold improvement over the unassembled parent strain (∼0.5 mg/g DCW), without any detectable effect on host growth kinetics. Collectively, these results establish SNARE-derived coiled-coil peptides as a biocompatible, modular, and programmable toolkit for spatially organizing biosynthetic enzymes, with broad applicability to metabolic engineering and biomanufacturing.</p>