Lipid Droplets As a New Platform for Protein Expression in Chlamydomonas Reinhardtii

Lipid droplets (LDs) are most often associated with biofuel production, as they are rich in neutral lipids, a desirable substrate for biodiesel production. The highly hydrophobic core of the LDs function as a storage compartment of variable small compounds, like carotenoids, sterols and terpenoids. They also have a physiological function in transporting lipids and proteins between membranes as well as storing them in the response to variable environmental stresses [1].

With the goal to sustainably produce high value products in the green algae Chlamydomonas reinhardtii we aim at production of heterologous proteins and assembling metabolic pathways on the surface of LDs by fusing enzymes to the anchoring proteins i.e. oleosins. Oleosins were used already for production of recombinant proteins in E. coli, yeasts and tobacco but not yet for expression of active enzymes and not yet in green eukaryotic algae. Oleosins are relatively small proteins with a highly hydrophobic proline-knot motif which anchor them to the LDs, and most probably, their insertion is driven solely by their physicochemical properties. By this strategy we will facilitate spatial organization of the enzymes and secretion of the hydrophobic products to the interior of the LDs [2].

First to show proper functioning of anchoring proteins we fused a fluorescent protein to different oleosins and successfully targeted these reporters to LDs in C. reinhardtii. The specific localization and bright fluorescence of these fusion proteins allows for detailed tracking of LDs and single-cell analysis of LD’s physiology. By use of image cytometry we can assess the number, size and distribution of LDs in every cell, and compare the distributions between strains containing different oleosin-reporter constructs at different growth stages. Next we will try to express cytochrome P450s from the dhurrin pathway from Sorhum bicolor by exchanging the native transmembrane region with the LD anchoring proteins. Enzymes from the dhurrin pathway form functional biosynthetic protein complexes, so-called metabolons, facilitating shuttling of intermediates between enzymes and increasing formation of the final product. Our aim is to co-localize enzymes from this pathway and investigate if the metabolon formation still takes place. This proof-of-concept will be a good starting point for assembly of more complicated biosynthetic pathways [3, 4].

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