Functional and Mechanistic Characterization of VacPT, a Novel <i>cis</i> -Prenyltransferase from <i>Vibrio alginolyticus</i>

cis -prenyltransferases (cPTs) play an essential role in natural rubber biosynthesis, yet their functional expression often depends on host-specific cofactors. In this study, we identified and characterized a novel cPT, designated Va cPT, from the marine bacterium Vibrio alginolyticus . Genomic analysis revealed that Va cPT is phylogenetically distinct and lacks associated biosynthetic gene clusters, suggesting functional independence. Heterologous expression of Va cPT in Escherichia coli resulted in the production of C 55 and C 70 polyisoprenoid products in vivo and in vitro, respectively. Enzymatic assays showed a strong substrate preference for farnesyl diphosphate (FPP), with kinetic parameters indicating higher affinity and catalytic efficiency compared to geranyl diphosphate (GPP). Structural modeling and docking analyses revealed a conserved active pocket within the dimeric interface, accommodating both FPP and isopentenyl diphosphate (IPP), stabilized by key residues (Arg24, Glu67, Asp144, Glu207, and Arg194) and Mg 2+ coordination. These findings establish Va cPT as a functionally independent cPT capable of medium-chain polyisoprenoid synthesis and highlight the potential of marine-derived enzymes for heterologous production of natural rubber precursors in prokaryotic systems.