First, we started from the „Protect“ side and asked if deletion of triacylglycerol (TAG) lipases would result in increased lipid accumulation. We made several single, double and a triple deletion of major yeast TAG lipases TGL3, TGL4 and TGL5 and analyzed the effect of these modifications on the lipid levels. For this, we grew the constructed yeast strains for 24 and 72 hours and measured the lipid levels by Nile red staining. Nile red enables specific detection of intracellular lipids that are in highly hydrophobic lipid droplets (LDs), and it does not stain the membrane lipids (Greenspan et al., 1985). Our experiments showed that deletion of tgl3Δ led to a considerable increase in lipid accumulation, and that this was further enhanced by triple tgl3Δ tgl4Δ tgl5Δ deletion (Fig 1, 2). These experiments show that the lipid accumulation can be increased by TAG lipase deletions, presumably by decreasing lipid turnover.
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.
To further increase lipid production via stabilization of TAGs, we overexpressed human perilipin gene PLIN3, which has been shown to promote lipid droplet (LD) assembly (Jacquier et al., 2013). For this, we constructed a plasmid that would express light-regulated transcription factor VP-EL222, and the PLIN3 gene from the VP-EL222-regulated promoter. This promoter is inactive in darkness, but is activated by blue light (Benzinger and Khammash, 2018). We transformed the plasmid to tgl3Δ tgl4Δ tgl5Δ zwf1Δ strain, grew the cells either under illumination or in darkness, and measured the lipid accumulation by Nile red staining. We observed substantially higher intracellular lipid levels in the culture that was exposed to light (Fig 3). This suggests that the light-activated expression of Plin3 results in higher lipid production.
Fatty acid synthesis is heavily dependent on NADPH supply, which in S. cerevisiae is regenerated in the pentose phosphate pathway (PPP), where a hexose sugar is oxidized and carbon is lost in the form of CO2. To maximize the production yield, we aimed to utilize a indium phosphide nanoparticle-yeast biohybrid system, where yeast cells could use light energy for NADPH regeneration (Guo et al., 2018). For this, we inactivated the oxidative part of PPP by ZWF1 deletion in the tgl3Δ tgl4Δ tgl5Δ strain and functionalized the cells with nanoparticles. Then, we measured the lipid production capabilities of these biohybrids compared to cells from a similar background without the nanoparticles. Nile red staining of lipid droplets revealed that the lipid levels were considerably higher in the biohybrid cells (Fig 3). This experiment shows that the light-harvesting nanoparticles can support lipid production in S. cerevisiae. Our experiments show that we have successfully improved lipid production by three different engineering approaches.
Figure 3. 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.
In order to demonstrate that microbial lipid production can be carried out in industrial settings, we decided to build a bioreactor that would reflect real production conditions. Our bioreactor, called “SANYA” provides users with the following key features:
More detailed description of SANYA’s internals and instructions can be found on the Hardware page.
Benzinger, D., and Khammash, M. (2018). Pulsatile inputs achieve tunable attenuation of gene expression variability and graded multi-gene regulation. Nat. Commun. 9, 1–10. Greenspan, P., Mayer, E.P., and Fowler, S.D. (1985). Nile red: A selective fluorescent stain for intracellular lipid droplets. J. Cell Biol. 100, 965–973. Guo, J., Suástegui, M., Sakimoto, K.K., Moody, V.M., Xiao, G., Nocera, D.G., and Joshi, N.S. (2018). Light-driven fine chemical production in yeast biohybrids. Science (80-. ). 362, 813–816. Jacquier, N., Mishra, S., Choudhary, V., and Schneiter, R. (2013). Expression of oleosin and perilipins in yeast promotes formation of lipid droplets from the endoplasmic reticulum. J. Cell Sci. 126, 5198–5209.
