Enhancing precursor supply and modulating metabolism to achieve high-level production of β-farnesene in Yarrowia lipolytica

β-Farnesene is a sesquiterpene commonly found in essential oils of plants, with applications spanning from agricultural pest control and biofuels to industrial chemicals. The use of renewable substrates in microbial cell factories offers a sustainable approach to β-farnesene biosynthesis. In this study, malic enzyme from Mucor circinelloides was examined for NADPH regeneration, concomitant with the augmentation of cytosolic acetyl-CoA supply by expressing ATP-citrate lyase from Mus musculus and manipulating the citrate pathway via AMP deaminase and isocitrate dehydrogenase. Carbon flux was modulated through the elimination of native 6-phosphofructokinase, while the incorporation of an exogenous non-oxidative glycolysis pathway served to bridge the pentose phosphate pathway with the mevalonate pathway. The resulting orthogonal precursor supply pathway facilitated β-farnesene production, reaching 810 mg/L in shake-flask fermentation. Employing optimal fermentation conditions and feeding strategy, a titer of 28.9 g/L of β-farnesene was attained in a 2 L bioreactor.