Fig. 3: Functional characterisation of EpSS-L1 and EpSS-L2 in S. cerevisiae.

AS. cerevisiae CEN.PK2-1C, wild type strain (-tHMG1) and truncated HMG-CoA reductase 1 -expressing strain (+tHMG1) were transformed with either Empty Vector (EV, pBEVY-L) or pBEVY-L constructs for overexpression of: A. thaliana Squalene Synthase (AtSS, GenBank accession BT003419.1), E. peplus Squalene Synthase-like1 (EpSS-L1, GenBank locus tag M5689_021805) and E. peplus Squalene Synthase-like2 (EpSS-L2, GenBank locus tag M5689_021806). Empty Vector (EV), A. thaliana Squalene Synthase (AtSS, GenBank accession BT003419.1), E. peplus Squalene Synthase-like1 (EpSS-L1, GenBank locus tag M5689_021805), E. peplus Squalene Synthase-like2 (EpSS-L2, GenBank locus tag M5689_021806) with and without truncated A. thaliana HMG-CoA reductase (HMGRt, GenBank accession J04537.1). S. cerevisiae cultures were extracted and analysed by LC-MS in parallel with E. peplus latex-purified peplusol standard. Extracted Ion Chromatograms (EIC) shown for m/z 427.4 showing peplusol (1) in extracts derived from cell pellets (B) Quantification of peplusol by LC-MS from the extracts shown on (A). Three independent transformants were grown in liquid cultures. Peplusol levels were quantified by LC-MS in: whole liquid cultures (grey bars), cell pellets (blue bars) and media (orange bars), against a standard curve generated from purified peplusol. n.d.: not detectable, Error bars: SEM (n = 3, where n is the number of biological replicates). Statistically significant (one-sided t-test) changes between EpSS-L1 overexpressed in -tHMG1 and +tHMG1 backgrounds indicated by asterisks for whole liquid cultures, cell pellets and media (*: p-value < 0.05, **: p-value < 0.01). C Quantification of squalene by GC-MS. Three independent transformants were grown in liquid cultures. Squalene levels were quantified by GC-MS in cell pellets from the strains shown in panel A. Error bars: SEM (n = 3, where n is the number of biological replicates). Statistically significant (one-sided t-test) differences between control (EV) and candidate genes indicated by asterisks (*: p-value < 0.05, **: p-value < 0.01). Source data are provided as a Source Data file.