Part:BBa_K3692002

PLIN3

Usage and Biology

PLIN3 is a gene that encodes perilipin - a protein which is involved both in generation of lipid droplets (LDs) and as well as covering of phospholipid monolayer of LDs.

PLIN3 is a human perilipin gene. However, it has been shown that the expression of this gene can increase yields of TAG by 28% in Sacharomyces cerevisiae (Teixeira et al., 2018). A potential reason for this can be that it stabilizes TAGs by improving LD assembly and the budding process.

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 1). 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 1). This shows that the light could also be used as an energy source to drive lipid synthesis.

Figure 1. 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.

References

• Teixeira, P. G., David, F., Siewers, V., & Nielsen, J. (2018). Engineering lipid droplet assembly mechanisms for improved triacylglycerol accumulation in Saccharomyces cerevisiae. FEMS Yeast Research, 18(6). https://doi.org/10.1093/femsyr/foy060
• 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. https://doi.org/10.1242/jcs.131896

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 369
  Illegal PstI site found at 218
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 369
  Illegal PstI site found at 218
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 369
  Illegal PstI site found at 218
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 369
  Illegal PstI site found at 218
  
• 1000
  COMPATIBLE WITH RFC[1000]