Engineering the oleaginous yeast Yarrowia lipolytica for β‐farnesene overproduction

Abstract

β‐farnesene is a sesquiterpenoid with various industrial applications which is now commercially produced by a Saccharomyces cerevisiae strain obtained by random mutagenesis and genetic engineering. We rationally designed a genetically defined Yarrowia lipolytica through recovery of L‐leucine biosynthetic route, gene dosage optimization of β‐farnesene synthase and disruption of the competition pathway. The resulting β‐farnesene titer was improved from 8 to 345 mg L‐1. Finally, the strategy for decreasing the lipid accumulation by individually and iteratively knocking out four acyltransferases encoding genes was adopted. The result displayed that β‐farnesene titer in the engineered strain CIBT6304 in which acyltransferases (DGA1 and DGA2) were deleted increased by 45% and reached 539 mg L‐1 (88 mg g‐1 DCW). Using fed‐batch fermentation, CIBT6304 could produce the highest β‐farnesene titer (22.8 g L‐1) among the genetically defined strains. This study will provide the foundation of engineering Y. lipolytica to produce other terpenoids more cost‐efficiently.

Recent Advances in the Biosynthesis of Farnesene Using Metabolic Engineering

“…Besides, the β-farnesene titer was greatly improved in Y. lipolytica by introducing carbon flux from fatty acid synthesis to the MVA pathway, and the maximum titer reached 22.8 g/L . These results also indicated that Y.…”

“…In Y. lipolytica, the α-farnesene titer increased 20.8-fold by introducing a carbon flux into the MVA pathway, and the titer reached 57.08 mg/ L. 40 Besides, the β-farnesene titer was greatly improved in Y. lipolytica by introducing carbon flux from fatty acid synthesis to the MVA pathway, and the maximum titer reached 22.8 g/L. 41 These results also indicated that Y. lipolytica has the potential to construct strains with a high farnesene yield. However, Y. lipolytica also has some shortcomings that restrict its development in farnesene biosynthesis.…”

“…In a recent study, acyltransferases (DAG and DAGO) were deleted to reduce competition between fatty acid synthesis and farnesene synthesis for acetyl-CoA. 41 The results showed that the fatty acid content of engineered Y. lipolytica decreased by about 70%, while the farnesene titer increased by about 55%. Notably, the fatty acid content of the strain further decreased after deleting the four acyltransferases (Dago, DGA2, LRO1, and ARE1), but the farnesene yield also decreased.…”

“…The reason for this is that the production of lipophilic farnesene is also affected by the reduction of the fatty acid content in the cell. 41 It is well-known that, in the MEP pathway, the first reaction is a transketolase-like condensation reaction of pyruvate and Dglyceraldehyde-3-phosphate (G-3-P). 90 The balance between these two precursors is a major factor limiting the isoprenoid yield in the MEP pathway.…”

YALIcloneNHEJ: An Efficient Modular Cloning Toolkit for NHEJ Integration of Multigene Pathway and Terpenoid Production in Yarrowia lipolytica

“…The Frozen-EZ Yeast Transformation II Kit was used for Y. lipolytica transformation ( Shi et al, 2021 ). A flow chart summarizing the construction of Y. lipolytica strains is shown in Figure 5B .…”