2.2 Strain construction
All strains constructed in this study are derived from the Y.
lipolytica strain ST6629, described in Holkenbrink et al. [16].
This strain has modifications related to decreased degradation of fatty
acids/alcohols and an increased pool of fatty acyl-CoAs. Y.
lipolytica strain ST4840, which was obtained from Agricultural Research
Service (NRRL, USA), served as a source for genomic DNA (gDNA). gDNA was
extracted using Quick-DNA™ Fungal/Bacterial Miniprep Kit (Zymo
Research). The entire list of the strains is provided in Table S5.
Yeast transformations were performed as described in Holkenbrink et al.
[19] with the modifications described below. Y. lipolyticastrains were streaked on Yeast Peptone Dextrose (YPD) plates (20 g/L
glucose, 10 g/L peptone, 10 g/L yeast extract, 15 g/L agar) and grown
for 24 h at 28°C. A small patch of biomass was taken with an inoculation
loop and re-streaked on YPD plates containing 0.7 g/L complete
supplement mixture (Formedium). After 24 h incubation at 28°C, the cells
were scraped off, resuspended in 1 mL 0.5 M sterile sucrose solution,
and centrifuged for 5 min at 3,000 g at room temperature. The
supernatant was discarded, the cells resuspended in 0.5 M sucrose
solution, and a volume corresponding to OD600 2.6 was
transferred to a sterile tube for transformation. Tubes were centrifuged
for 5 min at 3,000 g at room temperature, and the supernatant was
removed. 500-1,000 ng of gRNA plasmid and integration plasmid, which
previously was linearized with SmiI (Thermo Fisher Scientific) and
gel-purified prior transformation, were added to the pellet. The mixture
was resuspended in 100 µL of transformation mix (Supplementary Table S6)
and incubated at 39°C for 1 h. After the heat shock, the tubes were
centrifuged, the supernatant removed, and the pellet was resuspended in
500 µL liquid YPD medium. The cells were incubated for 2 h at 28°C 300
rpm shaking. The pellet was again collected by centrifugation,
resuspended in 100 µL of 0.5 M sterile sucrose solution, and plated on
YPD plates containing Hygromycin B (200 mg/L) (Carl Roth) or
Nourseothricin (250 mg/L) (Jena Bioscience) for selection. After 2-3
days of incubation at 28°C, single colonies appeared, which were tested
for correct integration by PCR using vector-specific primers and primers
complementary to genomic loci close to the integration site
(Supplementary Table S1).