Tunable nano‐oleosomes derived from engineered Yarrowia lipolytica

Abstract

Oleosomes are discrete organelles filled with neutral lipids surrounded by a protein-embedded phospholipid monolayer. Their simple yet robust structure, as well as their amenability to biological, chemical, and physical processing, can be exploited for various biotechnology applications. In this study, we report facile biosynthesis of functionalized oleosomes within oleaginous yeast Yarrowia lipolytica, through expression of oleosin fusion proteins. By fusing a cDNA clone of a sesame oleosin with either the coding sequence of a red fluorescent protein mCherry or a cellulosomal scaffolding protein cohesin from Clostridium cellulolyticum, these oleosin-fusion proteins were efficiently expressed and specifically targeted to and anchored on the surface of the oleosomes within the Y. lipolytica cells. The engineered oleosomes can be easily separated from the Y. lipolytica cell extract via floating centrifugation and both mCherry and cohesin domains are shown to be functional. Upon sonication, the engineered Yarrowia oleosomes exhibit a mean diameter of 200-300 nm and are found to be highly stable. The feasibility of co-displaying multiple proteins on the Yarrowia oleosomes was demonstrated by incubating cohesin-displaying oleosomes with different dockerin-fusion proteins. Based on this strategy, engineered oleosomes with both cell-targeting and reporting activities were created and shown to be functional. Taken together, the Yarrowia oleosome surface display system in which oleosin serves as an efficient membrane anchor motif shows great promise as a simple platform for creating tunable nanoparticles.

Yarrowia lipolytica strains and their biotechnological applications: how natural biodiversity and metabolic engineering could contribute to cell factories improvement

“…Prior to its use for subcellular compartment engineering, the technic of oleosome targeting had been developed by a research team of Hawaii University at Manoa (USA) for the purpose of designing tunable functional nanoparticles [87]. Surface display of different heterologous proteins on Y. lipolytica LB was obtained through fusion with a heterologous oleosin from plant and the resulting subcellular structure was fractioned through sonication into nano-oleosomes of tunable size.…”

“…Surface display of different heterologous proteins on Y. lipolytica LB was obtained through fusion with a heterologous oleosin from plant and the resulting subcellular structure was fractioned through sonication into nano-oleosomes of tunable size. The obtained armed oleosomes correspond to stable nanoparticles (200-300 nm of diameter) used as scaffold for protein display, which can be equipped with various functionalities and used for numerous biomedical applications such as biosensors, cell targeting or drug delivery [87]. This work has also brought the first proof-of-concept that co-expressing in Y. lipolytica different heterologous proteins fused with either a dockerin or a cohesin domain would lead to their in vivo self-assembly that, combined with their surface display on oleosomes, can generate functional nanofactories applicable to various biotechnological purposes [87].…”

“…They can be retained in the ER if a C-terminal Lys-Asp-Glu-Leu (KDEL) amino acid sequence is present, or targeted to the cell peroxysomes if a C-terminal tripeptidic peroxisome targeting signal (PTS) is added, the amino acid sequences Ala-Lys-Ile (AKI) or Ser-Lys-Leu (SKL) being the more effective ones in Y. lipolytica [45,83,219]. Recombinant proteins can also be addressed to LB and oleosomes through transcriptional fusion of their ORF with the C-terminal amphipathic domain of a heterologous oleosin, a structural protein associated with the single layer membrane of plant oil bodies [83,87]. This oleosome targeting strategy was developed at first for the design of tunable functional nanoparticles that could be assembled in Y. lipolytica cells for various biotechnological application [87], as described in Section 2.3.5.…”

Synthetic biology tools for engineering Yarrowia lipolytica

“…Among the intracellular organelles, Y. lipolytica peroxisomes can be targeted by the peroxisomal targeting signal (PTS) domain, as the tripeptides AKI or SKL can be fused to the C-terminus of the protein ( Haddouche et al, 2010 ; Xue et al, 2013 ). Y. lipolytica lipid bodies can be targeted by fusing the protein to the C-terminal domain of oleosin, a structural protein embedded in the phospholipid membrane of plant oleosomes ( Han et al, 2013 ). The nucleus can be targeted by fusing the well-known viral SV40 nuclear localization sequence (NLS) (PKKKRKV) to the protein (e.g., for efficiently targeting Cas9; Schwartz et al, 2016 ).…”

Yarrowia lipolytica as an Oleaginous Cell Factory Platform for Production of Fatty Acid-Based Biofuel and Bioproducts

“…Triglycerides (TAG) and steryl esters (SE) are the major cellular lipids synthesized from sterol, glycerol-3-phosphate, and acyl-CoA. These lipid compounds comprise the hydrophobic core of the dynamic storage compartment called oleosome or lipid bodies (LB) (Han et al, 2013). LB can also accommodate FFA.…”