Staphyloxanthin (STX) is a saccharolipid derived from a carotenoid in <Staphylococcus aureus> involved in oxidative-stress tolerance and antimicrobial peptide resistance. In this work, a targeted metabolomics and biophysical study was carried out on native and knock-out S. aureus strains to investigate the biosynthetic pathways of STX and related carotenoids. Identification of 34 metabolites at different growth phases (8, 24 and 48h), reveal shifts of carotenoid populations during progression

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... wards stationary phase. Six of the carotenoids in the STX biosynthetic pathway and three menaquinones (Vitamin K2) were identified in the same chromatogram. Furthermore, other STX homologues with varying acyl chain structures reported herein for the first time, which reveal the extensive enzymatic activity of CrtO/CrtN. Fourier Transform infrared spectroscopy show that STX increases acyl chain order and shifts the cooperative melting of the membrane indicating a more rigid lipid bilayer. This study shows the diversity of carotenoids in S. aureus, and their influence on membrane biophysical properties.