Fig. 4: In vivo evaluation of lipid-dependent migration and storage in LDs.

A The biosynthesis pathway of three non-saponifiable lipids, squalene, β-carotene, and zeaxanthin. Squalene was produced by overexpressing native squalene pathway genes (thmg1 and erg20). β-carotene and zeaxanthin were produced via heterologous biosynthetic pathways upon expression of genes obtained from Pantoea agglomerans (crtE, crtB, crtI, crtY, and crtZ). B Quantitative measurements of squalene and zeaxanthin produced by designer yeast cells. Structurally flexible squalene and rigid zeaxanthin showed a completely different storage-dependence pattern; the increase in squalene and zeaxanthin storage was significantly correlated with the increase in the total volume (size) and net surface area (number) of the LDs, respectively. C Localization of rigid β-carotene at the LD surface. Rod-like β-carotene showed a storage pattern in LDs very similar to that of zeaxanthin. Using confocal microscopy, we directly observed that β-carotene (red) was favorably adsorbed into all kinds of membranes, including the LD surface (green). Yeast cells were grown in defined minimal media with 2% glucose at 30 °C for 24 h. We note that β-carotene was inherently fluorescent (excitation at 450 nm and emission at 600 nm), and LDs were stained with the BODIPY fluorescent dye (excitation at 488 nm and emission at 500 nm). For production of target lipids, yeast cells were grown in defined minimal media with 2% glucose in the presence (+) or absence (−) of 10 µg/mL terbinafine at 30 °C for 144 h. The inhibition of squalene epoxidase (Erg1) by terbinafine was helpful to further increase production of squalene, zeaxanthin, and β-carotene, but it did not alter the distinctive storage patterns of the flexible and rigid lipids. All data represent the mean of biological triplicates, and error bars indicate the standard deviation. An asterisk (*) indicates that the value is significantly different (P < 0.05) from that for the respective control cells. Significance (P-value) was evaluated by two-sided t-test. IPP isopentenyl pyrophosphate; DMAPP dimethylallyl diphosphate; GPP geranyl pyrophosphate; FPP farnesyl pyrophosphate; GGPP geranylgeranyl pyrophosphate. Scale bar, 5 μm. Source data are provided in the Source Data file.