Fig. 2. Signal peptide-guided tSPFs selectively transport loaded squalene to the extracellular medium.

A Schematic illustration of squalene production and recovery by our metabolite trafficking strategy. An SQ03-INO2 yeast strain (SQ), capable of squalene overproduction, was engineered to overexpress tSPF derivatives tagged with export signal peptides (left). Unlike conventional methods, such as cell disruption, which rely on multiple steps for squalene extraction (right, top), the signal peptide-tagged tSPF derivatives enable continuous flow production of squalene (right bottom) in an energy-efficient and cost-effective manner. B High-performance liquid chromatograms of squalene secreted by tSPF derivatives. At the same retention time of the squalene standard (red), signal peptide-tagged tSPFs (Suc2-tSPF: navy; Pho5-tSPF: green; and MFα-tSPF: purple) yielded distinct peaks due to extracellular squalene secretion. When tSPF was neither tagged with signal peptides (pink) nor expressed in yeast (blue), only negligible peaks were detected. C Quantitative measurements of secreted and accumulated squalene. SQ strains included each export signal peptide and tSPF genes (left). Extracellular and intracellular squalene (middle and right) was quantified by collection of the dodecane phase and disruption of the harvested cells, respectively. D Western blot analysis of cell lysates (top) and culture supernatants (bottom). With signal peptides, most tSPFs migrated into the culture medium, but without them, the tSPF stayed in the cell. E Differential interference contrast (DIC) and confocal fluorescence microscopy images of yeast cells that overexpressed tSPF derivatives fused with a green fluorescent protein (GFP). Owing to extracellular secretion, fluorescent GFP-fused Suc2-tSPF and Pho5-tSPF were not accumulated inside the cells. In comparison, MFα-tSPF was not efficiently secreted from the cell; however, the non-tagged tSPF was completely reserved inside the cells. Scale bar: 5 μm. F Semi-continuous fermentation system using Suc2-tSPF/SQ. With a continuous nutrition supply, yeast cells with Suc2-tSPF enable sustainable squalene production and secretion. During semi-continuous culture, the growth medium was replenished every 3 days (left), and the levels of intra- and extracellular squalene were monitored for 15 days (right). All data represent the mean of biological triplicates, and error bars indicate the standard deviation. Source data are provided in the Source Data file.