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Sulfonolipids (SoLs) are a unique class of sphingolipids featuring a sulfonate group compared to other sphingolipids. However, the biological functions and biosynthesis of SoLs in human microbiota have been poorly understood. Here, we report the discovery and isolation of SoLs from a human opportunistic pathogen Chryseobacterium gleum DSM16776. We show for the first time the pro-inflammatory activity of SoLs with mice primary macrophages. Furthermore, we used both in vivo heterologous expression and in vitro biochemical reconstitution to characterize two enzymes, cysteate synthase and cysteate fatty acyltransferase, that are specifically involved in the biosynthesis of SoLs rather than other sphingolipids. Based on these two SoL-specific enzymes, our bioinformatics analysis showed a wider distribution of SoL biosynthetic genes in microbes that had not been reported as SoL producers. We selected four …