Strains and Medium
After replacing PCAT1 promoters of α-farnesene
high-producing strain P. pastoris X33-30 with promoter
PGAP, strain X33-30*, which was reconstructed the
carbon’s metabolic pathways in P. pastoris X33 was used as a host
for gene modification. The above P. pastoris strains were
cultivated in YPD medium at 30°C, and P. pastoris engineered
recombinant strains were screened with 100 mg/L zeocin or 500 mg/L
geneticin, recovery of selectable markers by Cre/LoxP system. The rich
YPD medium containing 20 g/L
glucose, 20 g/L peptone, and 10 g/L yeast extract. The medium YPDA for
inducing Cre enzyme expression contains: 20 g/L L-galactose, 10 g/L
yeast extract and 20 g/L peptone. E. coli JM109 was used for gene
cloning with antibiotics (25 mg/L of zeocin, 50 mg/L of kanamycin or 100
mg/L of ampicillin).
Construction
of plasmids and strains
Some of the constructed strains and recombinant plasmids in this study
were listed in Table 1 and Table 2 respectively. The designed primers
were listed in Table S1 of Supporting Information.
The integration site of the strain
was the PGAP promoter site or the his4 site. The
specific strain construction procedures were included in the Supporting
Information. The LoxP sites in the Cre/LoxP system were mutated into
Lox71 and Lox66 sites, respectively, to prevent repeated cleavage and
recombination by Cre enzyme. The details of DNA manipulation and
transformation were described in
” Supporting Information”.
Shake
flask culture conditions and biomass analysis
Preserved strains were cultured in YPD medium at 30 ° C for activation.
Then the appropriate amount of activated strain was put into 10 ml
liquid medium overnight to become seed medium. Initial
OD600 after inoculation with 50 mL YPD fermentation
medium was 0.15, adjusted to pH 6.0 with 100 mM/L potassium phosphate
buffer, the upper layer was overlaid with 10% n-dodecane, and the
fermentation was completed after 72 h in a reciprocating shaker at 30 °
C with 100 rpm. Optical density (OD) of P. pastoris cells was
measured at 600 nm with a spectrophotometer. Dry cell weight was
measured as described by Tomas et al11.
Quantification of
α-farnesene
The fermentation broth was
centrifuged at 12,000 rpm for 10 minutes. Then the upper layer of
n-dodecane liquid was filtered, and the yield of a-farnesene could be
measured. The detection method of a-farnesene is as described by Liu et
al3. The standard α-farnesene, antibiotics and
chemicals were purchased from Sigma (Sigma Aldrich, USA).