Mitochondrial Engineering of Yarrowia lipolytica for Sustainable Production of α-Bisabolene from Waste Cooking Oil

Abstract

Metabolic engineering of yeasts for terpenoid production has mostly focused on the cytoplasm, whereas harnessing their organelles as subcellular factories has been overlooked. Herein, the farnesyl diphosphate synthetic pathway and α-bisabolene synthase were compartmentalized into the oleaginous yeast Yarrowia lipolytica's mitochondria to enable high-level α-bisabolene production. Through comprehensive metabolic engineering approaches, we exploited the potential and capability of the mitochondria as a subcellular factory to achieve 257.4 mg/L of α-bisabolene production from glucose. By combining mitochondrial and cytoplasmic engineering, we further boosted the α-bisabolene titer to 765.1 mg/L by utilizing waste cooking oil as the sole carbon source. Finally, the α-bisabolene titer of the resulting strain reached 1058.1 mg/L in a 5 L bioreactor, which is the highest titer in the engineered Y. lipolytica cell factory reported to date. Overall, our study has provided valuable insights into the mitochondrial engineering of Y. lipolytica for sustainable and green production of valuable compounds.

Enhancing Trans-Nerolidol Productivity in Yarrowia lipolytica by Improving Precursor Supply and Optimizing Nerolidol Synthase Activity

“…Zhao et al found that the single overexpression of ERG19 could improve the bisabolene production in Y. lipolytica strain harboring only the bisabolene synthase . Guo et al found that the titers of (+)-valencene and (+)-nootkatone in Y.…”

“…5 For example, harnessing the organelle-derived acetyl-CoA was proven to be able to enhance acetyl-CoA availability and successfully increased the titers of α-humulene and amorpha-4,11-diene to 21.7 g/L and 427 mg/L in Yarrowia lipolytica and S. cerevisiae with the combination of cytoplasmic and peroxisomal or mitochondrial engineering. 6,7 Besides, integration of strengthening the MVA pathway and repressing the com-petitive sterol biosynthesis strongly supported the FPP flux toward target sesquiterpene production in S. cerevisiae or other yeasts, such as trans-nerolidol, 8−10 amorphadiene, 11,12 bisabolene, 13 farnesene, 14,15 α-santalene, 16,17 caryophyllene, 18 and longifolene. 19 The rate-limiting steps identified in the above studies include almost all of the genes related to the MVA pathway but varied with the specific sesquiterpene and yeast host.…”

“…43 Zhao et al found that the single overexpression of ERG19 could improve the bisabolene production in Y. lipolytica strain harboring only the bisabolene synthase. 13 Guo et al found that the titers of (+)-valencene and (+)-nootkatone in Y. lipolytica were correspondingly increased by 2.1-and 1.8-fold in the engineered strains with the ERG20 overexpression. 44 Spatial clustering of enzymes could optimize substrate channeling for the efficient transfer of metabolic pathway intermediates from one active site to another in sequential reactions.…”

“…Due to these outstanding traits, Y. lipolytica has been engineered for efficiently producing terpenoid compounds and fatty acid-derived chemicals. ,− ,,,− However, the cytosolic acetyl-CoA in Y. lipolytica is mainly directed toward lipid biosynthesis instead of the MVA pathway .…”

Combining Metabolic Engineering and Lipid Droplet Storage Engineering for Improved α-Bisabolene Production in Yarrowia Lipolytica

“…These results are in accordance with a previous study conducted by Cao et al In our previous study, we demonstrated that the ABC-G1 transporter fromGrosmania clavigera effectively mitigated cytotoxicity and increased the overall production titer of α-bisabolene in engineered Y. lipolytica. , Here, we overexpressed the ABC-G1 transporter in strain Po1gαB1, and the intracellular α-bisabolene production was decreased from 5.82 to 1.65 mg/L (Figure S4). According to Figure B, strain Po1gαB2 produced approximately 97.5 mg/L of α-bisabolene after 5 days of fermentation in YPD medium, representing a 3.9-fold increase in α-bisabolene titer compared to the control strain Po1gαB0 (20.1 mg/L) (Figure B).…”