We started by inhibiting the degradation of triacylglycerols (TAGs), as this has been shown previously to result in increased TAG accumulation (Ferreira et al., 2018). We made single, double, and triple deletion of TAG lipases TGL3, TGL4 and TGL5 and measured the effect of these modifications on lipid accumulation by Nile red staining of yeast. We found that the triple deletion resulted in highest lipid levels, thus we decided to continue with this strain for further modifications (Fig 1, 2).
Figure 1. Nile red staining shows build-up of lipids in LDs upon TAG lipase deletions. Microscopy images showing the cells in brightfield image and the fluorescent signal of lipids stained with Nile red. The cells are from 24 hour time point after inoculation.
Figure 2. TAG lipase deletions result in greatly increased lipid accumulation in LDs. (A, B) Plots showing the mean Nile red fluorescence intensities in single cells from indicated strains. The samples were analyzed at 24 hour time point (A) and 72 hour time point (B). The bars show median and its 95% confidence intervals. ****, **, *, and ns note p-values <0.0001, <0.01, <0.05, and >0.05, respectively, of pair-wise comparisons with wild-type using Mann-Whitney U test.
In addition to increasing the stability of TAGs, we aimed to maximize the carbon flux to final product synthesis. An innovative solution for this has been suggested by Guo et al., 2018 (Guo et al., 2018), who functionalized yeast cells with indium phosphide nanoparticles to give yeast an ability to utilize solar energy for NADPH regeneration. As fatty acid synthesis is highly dependent on NADPH availability (Sheng and Feng, 2015), we hypothesized that the indium phosphide nanoparticles could be very beneficial to support the lipid production. To avoid carbon loss via NADPH regeneration, the oxidative part of the pentose phosphate pathway must be inactivated. This has been done by deletion of ZWF1 (Guo et al., 2018). Thus, we deleted ZWF1 in the tgl3Δ tgl4Δ tgl5Δ strain.
In S. cerevisiae, lipids generally accumulate in the stationary phase (Werner-Washburne et al., 1993). Also, increased lipid synthesis can burden the cell metabolism and decrease cell growth. For an efficient cultivation, we wanted to separate growth and production phases. Therefore, we find it important to also check how the different genetic modifications affect the growth rate. For this, we performed growth rate assays, which revealed that although TAG lipase deletions did not have a marked effect on cell growth, the ZWF1 deletion did (Fig 3). Based on this result, we think that our strain could be further improved by making a conditional shutoff version of ZWF1, instead of a conventional deletion. Interestingly, recent studies have found several efficient methods for inducible protein depletion, including light-dependent possibilities (Deng et al., 2020).
Figure 3. Viability assay shows decreased growth rate of zwf1Δ strain.
Not to burden the cell with lipid production during the growth phase, we wanted to create an inducible metabolic switch. We chose to use light to control this switch, as in comparison with other known inductors, light is relatively cheap, easily controllable, and does not require addition of extra compounds to the growth medium. Based on published data, we planned to overexpress three genes: PAH1 and DGA1, which encode for enzymes in the TAG synthesis pathway, and human perilipin gene PLIN3. Plin3 has been shown to promote lipid droplet assembly, which stabilizes TAGs and enables their higher accumulation (Jacquier et al., 2013). For the inducible overexpression, we used light-regulated transcription factor VP-EL222, which is sensitive to the level on illumination and has very low background activity (Benzinger and Khammash, 2018). To mimimize the number of genetic modification steps to make the production strain, we constructed an overexpression vector that also contained the VP-EL222 transcription factor. We transformed the tgl3Δ tgl4Δ tgl5Δ zwf1Δ strain cells with a plasmid containing the PLIN3 expression cassette and analyzed the lipid accumulation in these cells when grown with or without illumination (Fig 4). These experiments revealed that the lipid levels in LDs were increased upon illumination, indicating that the part we constructed has a beneficial effect on lipid production in yeast. Also, we observed an increased lipid build-up in cells that were functionalized with the indium phosphide nanoparticles and were grown under illumination (Fig 4). This shows that the light could also be used as an energy source to drive lipid synthesis.
Figure 4. Light-harvesting nanoparticles and induced expression of perilipin greatly promote lipid accumulation. (A) Microscopy images of exemplary cells showing the accumulation of lipids in LDs detected by Nile red staining. The strains based on tgl3Δ tgl4Δ tgl5Δ zwf1Δ background with PLIN3 expression plasmid or indium phosphide (InP) nanoparticles were grown with or without illumination. (B) Plot showing the average fluorescence intensities of Nile red staining. The bars show median values and its 95% confidence intervals. **** indicates p-value <0.0001 in pair-wise comparisons with PVPEL222-PLIN3 grown without illumination by Mann-Whitney U test.
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