Engineering Yarrowia lipolytica for the sustainable production of β-farnesene from waste oil feedstock

Abstract

Background β-Farnesene is a sesquiterpene with versatile industrial applications. The production of β-farnesene from waste lipid feedstock is an attractive method for sustainable production and recycling waste oil. Yarrowia lipolytica is an unconventional oleaginous yeast, which can use lipid feedstock and has great potential to synthesize acetyl-CoA-derived chemicals. Results In this study, we engineered Y. lipolytica to produce β-farnesene from lipid feedstock. To direct the flux of acetyl-CoA, which is generated from lipid β-oxidation, to β-farnesene synthesis, the mevalonate synthesis pathway was compartmentalized into peroxisomes. β-Farnesene production was then engineered by the protein engineering of β-farnesene synthase and pathway engineering. The regulation of lipid metabolism by enhancing β-oxidation and eliminating intracellular lipid synthesis was further performed to improve the β-farnesene synthesis. As a result, the final β-farnesene production with bio-engineering reached 35.2 g/L and 31.9 g/L using oleic acid and waste cooking oil, respectively, which are the highest β-farnesene titers reported in Y. lipolytica. Conclusions This study demonstrates that engineered Y. lipolytica could realize the sustainable production of value-added acetyl-CoA-derived chemicals from waste lipid feedstock.

High-Efficiency Multiplexed Cytosine Base Editors for Natural Product Synthesis in Yarrowia lipolytica

“…We hypothesized GPD1, CIT2, and MLS knockouts would increase malonyl-CoA accumulation by increasing acetyl-CoA availability, whereas DGA1 and DGA2 are targeted to divert the malonyl-CoA flux toward naringenin production from lipids. We observed that the knockouts of DGA1 and DGA2 did not increase naringenin production, contradicting the previous work on β-farnesene production . We hypothesize that it is because the knockouts were performed on a strain engineered to possess a high acetyl-CoA flux.…”

“…Several knockouts in both the tyrosine and malonyl-CoA pathways were designed based on inference from the literature. Double knockouts of DGA1 and DGA2 increased β-farnesene production by 56% as the lipid content was reduced to 8.72% from 26% . The addition of fatty acid inhibitor cerulenin increased naringenin production by 31.2% in engineered strains, pointing to the significance of fatty acid downregulation to increase malonyl-CoA-based molecules.…”

“…Double knockouts of DGA1 and DGA2 increased β-farnesene production by 56% as the lipid content was reduced to 8.72% from 26%. 43 The addition of fatty acid inhibitor cerulenin increased naringenin production by 31.2% in engineered strains, 8 pointing to the significance of fatty acid downregulation to increase malonyl-CoA-based molecules. GPD1 overexpression was combined with DGA1 overexpression to increase lipid production by 12% compared to overexpressing DGA1.…”

“…We observed that the knockouts of DGA1 and DGA2 did not increase naringenin production, contradicting the previous work on β-farnesene production. 43 We hypothesize that it is because the knockouts were performed on a strain engineered to possess a high acetyl-CoA flux. We also observed that knockouts of MLS1 and CIT2 increased naringenin production, as hypothesized based on prior work to produce crocetin in S. cerevisiae.…”

Carotenoid Cleavage Dioxygenase 1 and Its Application for the Production of C13-Apocarotenoids in Microbial Cell Factories: A Review

“…In recent years, oleaginous microbial hosts, especially the nonconventional yeast Yarrowia lipolytica , have been successfully developed for the production of various terpenoid molecules such as farnesene, amorphadiene, squalene, lycopene, and β-carotene. − When compared to conventional model microbial hosts, such as E. coli and S. cerevisiae , Y. lipolytica was characterized by great advantages, including a strong pool of precursor acetyl-ConA, natural storage environment for hydrophobic compounds, numerous lipid droplets, and sufficient cofactor . In addition, Y. lipolytica is also generally recognized as safe (GRAS) by the American Food and Drug Administration (FDA) …”