β-Carotene is a yellow–orange–red pigment used in food, cosmetics and pharmacy. There is no commercial yeast-based
process for β-carotene manufacturing. In this work, we engineered the baker’s yeast Saccharomyces cerevisiae by expression
of lipases and carotenogenic genes to enable the production of β-carotene on hydrophobic substrates. First, the
extracellular lipase (LIP2) and two cell-bound lipases (LIP7 and LIP8) from oleaginous yeast Yarrowia lipolytica were expressed
either individually or in combination in S. cerevisiae. The engineered strains could grow on olive oil and triolein as the sole
carbon source. The strain expressing all three lipases had ∼40% lipid content per dry weight. Next, we integrated the genes
encoding β-carotene biosynthetic pathway, crtI, crtYB and crtE from Xanthophyllomyces dendrorhous. The resulting engineered
strain bearing the lipases and carotenogenic genes reached a titer of 477.9 mg/L β-carotene in yeast peptone dextrose (YPD)
medium supplemented with 1% (v/v) olive oil, which was 12-fold higher than an analogous strain without lipases. The
highest β-carotene content of 46.5 mg/g DCW was obtained in yeast nitrogen base (YNB) medium supplemented with 1%
(v/v) olive oil. The study demonstrates the potential of applying lipases and hydrophobic substrate supplementation for the
production of carotenoids in S. cerevisiae.