Abstract

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 VacPT, from the marine bacterium Vibrio alginolyticus. Genomic analysis revealed that VacPT is phylogenetically distinct and lacks associated biosynthetic gene clusters, suggesting functional independence. Heterologous expression of VacPT in Escherichia coli resulted in the production of C55 and C70 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 Mg2+ coordination. These findings establish VacPT 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.