A Farnesyl Pyrophosphate Synthase from Apostichopus japonicus Is Involved in Saponin Biosynthesis and Regulated by Immune Processes

Abstract

Apostichopus japonicus is one of the few animals that can synthesize saponins. Present understanding of saponin biosynthesis is often limited to genome analysis, leaving the precise mechanism still unclear. This study examined the potential role of a gene annotated as farnesyl pyrophosphate synthase (FPS) in saponin biosynthesis of A. japonicus. We amplified its complete cDNA (denoted as AjFPS) and produced its recombinant protein (rAjFPS) through rapid amplification of cDNA ends (RACE) and Escherichia coli expression system, respectively. RNA interference and correlation analysis of AjFPS transcriptional expression level and saponin content were conducted to evaluate the role of AjFPS in saponin biosynthesis. A pull-down assay was used to preliminarily explore its regulatory role in saponin biosynthesis. The complete cDNA sequence of AjFPS was shown to be 3,006 bp in length. The inferred amino acid sequence harbored two conserved aspartate-rich sites (DDXXD). Analysis of the amino acid sequence using phylogenetics showed that AjFPS was more similar to FPSs found in saponin-producing marine organisms rather than those in non-saponin-producing ones or plants. The knockdown of AjFPS resulted in a remarkable decline in saponin content. The temporal expression of AjFPS was significantly correlated with the variation in saponin content after Vibrio splendidus challenge. The pull-down analysis illustrated that AjFPS interacted directly or indirectly with diverse immune-related factors associated with pathogen recognition and destruction and protection against oxidative stress. Combined, these results suggested that AjFPS is involved in saponin synthesis, and this process in A. japonicus is likely modulated by immune processes, with AjFPS serving as a regulatory node. These findings will promote a deeper understanding of saponin synthesis and immune regulation as well as contribute to the improvement of saponin quality traits in cultured A. japonicus.