OUR MOTIVATION

Palm oil is the most widely used vegetable oil in the world, with a production of around 60 million tons per year. Its mass production has destructive consequences for the environment. Indeed, forests are massively being replaced with palm plantations in countries like Indonesia or Malaysia, causing the extinction of many iconic species such as the Orangutan. Therefore, it is urgent to propose alternatives to palm oil production.

OUR PROJECT

Our project aims to develop a new chassis for lipid production with a green microalga named Chlamydomonas reinhardtii. As a proof-of-concept, we aim to produce the main components of palm oil, for instance palmitic acid and oleic acid.

We have taken our inspiration from a strategy that has successfully worked in plants: “Push, Pull and Protect”¹. It involves optimizing the flux of carbon into TAG (storage form of lipids) by increasing the fatty acid synthesis (Push), TAG assembly (Pull) and lipid turnover (Protect).

For this reason, we identified three important enzymes. The first one is FAT (fatty acid thioesterase) which plays a role in the export of fatty acids from the chloroplast to the endoplasmic reticulum. The second one is LPAAT (lysophosphatidic acid acyltransferase) that is important for fatty acids assembly into TAG. The last one is DGAT (diacylglycerol acyltransferase) that seems to be overexpressed in the mesocarp of the african oil palm ². So on one hand, to produce palmitic acid (C16:0), we want to express FAT-B2, LPAAT-A and DGAT-1-2 from African oil palm Elaeis guineensis in Chlamydomonas reinhardtii using the Golden Gate Modular Cloning (MoClo) technology, based on Type IIS restriction enzymes. On the other hand, to produce oleic acid (C18:1), we want to express FAT-A, LPAAT-A and DGAT-1-2 in C. reinhardtii. Finally, to enhance TAG production, we will grow C. reinhardtii in nitrogen starvation conditions.

In parallel of our main project, we implemented the HiBiT technology in the MoClo kit by standardizing and integrating it into the C. reinhardtii MoClo kit. This 11 amino acid tag developed by Promega allows for a quick and effective measurement of our enzymes expression. The HiBiT tag is essentially one part of the NanoBiT® enzyme. When the HiBiT-tagged protein is incubated with the LgBiT, which is the other part of the NanoBiT, and its substrate, a functional NanoBiT will be assembled and emit a measurable and quantifiable luminescence signal.

OUR RESULTS

HiBiT validation in Chlamydomonas reinhardtii

This result points out that our HiBiT tags (BBa_K2909000 and BBa_K2909001) is expressed and functional in our clones in the presence of NO3. Therefore, we have successfully implemented the HiBiT technology in the MoClo kit by standardizing and integrating it into the C. reinhardtii MoClo kit.

LPAAT-A and DGAT-1-2 expression validation in Chlamydomonas reinhardtii

These second and third graphes show the measurement of nanoluciferase activity in different clones transformed with the following pCMM devices:

pCMM-6 : HiBiT-DGAT-1-2_HygroR E. guineensis (BBa_K2909011)
pCMM-7 : DGAT-1-2-HiBiT_HygroR E. guineensis (BBa_K2909012)
pCMM-8 : HiBiT-LPAAT-A_HygroR E. guineensis (BBa_K2909009)
pCMM-9 : LPAAT-A-HiBiT_HygroR E. guineensis (BBa_K2909010)
pCMM-14 : HiBiT-DGAT-1-2_HiBiT-LPAAT-A_HygroR E. guineensis (BBa_K2909013)
pCMM-15 : DGAT-1-2-HiBiT_LPAAT-A-HiBiT_HygroR E. guineensis (BBa_K2909014)

These results point out that our enzymes LPAAT-A (BBa_K2909002) and DGAT-1-2 (BBa_K2909003) are expressed in our clones in the presence of NO3. Therefore, our engineered C. reinhardtii strains successfully express the LPAAT-A and DGAT1-2 enzymes.

Fatty acids analysis

This figure shows thin-layer chromatography (TLC) analysis of lipid droplets of two clones transformed with the following pCMM devices (each circle corresponds to an area revealed by the UV light):

pCMM-7 : DGAT-1-2-HiBiT_HygroR E. guineensis
pCMM-15 : DGAT-1-2-HiBiT_LPAAT-A-HiBiT_HygroR E. guineensis

We can see that for the pCMM7-3 clone there is no DAG area compared to the WT clone. We can suppose that in this clone the DGAT enzyme has turned DAGs into TAGs. This result may suggest that we successfully increase TAG assembly (pull) by expressing the african oil palm DGAT-1-2 in C. reinhardtii. This result is very promising and more experiments will be needed to characterize and validate our engineered strains.

CONCLUSION

We built a project in a photosynthetic chassis, Chlamydomonas reinhardtii, in order to solve an actual environment issue. During this project, we have successfully implemented the HiBiT technology into the C. reinhardtii MoClo kit. This tool will allow for a quick and effective measurement of protein expression in C. reinhardtii for laboratories and future iGEM teams that will want to work with this chassis. Moreover, we successfully expressed the LPAAT-A and DGAT1-2 enzymes in C. reinhardtii and submitted these parts to the Registry. Finally, after the analysis of the lipid droplets of our engineered microalga, we obtained promising results that may suggest a future development of C. reinhardtii as a new chassis for lipid production.

For more details about Chlamydomonas reinhardtii, go on our Chlamy Guide :

THE CHLAMY GUIDE

Vanhercke, T. et al. Metabolic engineering of biomass for high energy density: oilseed-like triacylglycerol yields from plant leaves. Plant Biotechnol. J. 12, 231–239 (2014).
Dussert, S. et al. Comparative transcriptome analysis of three oil palm fruit and seed tissues that differ in oil content and fatty acid composition. Plant Physiol. 162, 1337–1358 (2013).

Team:Sorbonne U Paris/Plant