Fig. 3: Extension of our metabolite trafficking strategy for the secretion of other terpenes.

A The biosynthetic pathway of β-carotene production. Compared to the squalene biosynthesis pathway, this pathway further involves the expression of three constitutive genes, namely crtE, crtYB, and crtI. B Quantitative measurements of secreted and accumulated β-carotene produced by the engineered strains that overexpress Suc2-tSPF. Yeast cells were grown in a defined minimal medium with 2% (w/v) glucose and 10% (v/v) dodecane at 30 °C for 144 h. All data represent the mean of biological triplicates, and error bars indicate the standard deviation. C High-performance liquid chromatograms of β-carotene secreted by Suc2-tSPF. Compared to the β-carotene standard (red), overexpression of Suc2-tSPF (navy) resulted in a distinct peak at the same retention time, thus validating the extracellular secretion of β-carotene. In contrast, non-tagged tSPF (pink) failed to transport β-carotene outside cells. D Colorimetric determination of secreted β-carotene, an orange-colored terpenoid. After 144 h of cultivation, the expression of Suc2-tSPF resulted in a visible color change of dodecane due to extracellular β-carotene secretion. However, when only tSPF or no carrier protein was expressed, the dodecane phase showed faint coloration. E The binding promiscuity of tSPF towards a wide range of hydrophobic terpenes produced by microbial cells. The binding energy between SPF and each terpene predicted by molecular docking with AutoDock Vina in PyRx varied from −10.7 to −6.1 kcal/mol, which was comparable to the binding energy between SPF and squalene. Source data are provided in the Source Data file.