2. Experimental Section
2.1. Strains, plasmid, primers, and
chemicals
This study lists all stains, recombinant plasmids, and primers in
Supplementary Table1 and 2, respectively. Herein, strain
po1g[20] was chosen as the starting strain.
Chemicals used in this study, including glucose,
(NH4)2SO4, and YNB
(yeast nitrogen base without
(NH4)2SO4 and amino
acids), were all purchased from Sangon Biotech Co., Ltd (Shanghai,
China). The Nano-Glo® Luciferase Assay System kit was purchased from
Promega (Catalog: #N1120), and CSM-Leu powder was purchased from
Sunrise Science Products (Catalog: #1001-100).
2.2. Construction of the luciferase reporter vectors
The reporter vectors were constructed based on the plasmid pYLXP’, and
NanoLuc luciferase is encoded by the geneNluc .[19] Firstly, the chassis plasmid
pYLXP’-Nluc was obtained by the Gibson Assembly method, using the
gene Nluc fragment (PCR-amplified by primers Nluc-F and Nluc-R
from pYLXP’-PTEF-Nluc ) and linearized pYLXP’
(digested by SnaBI and KpnI ). Then, plasmid
pYLXP’-Nluc was further digested by SnaBI andAvrII , giving linearized pYLXP’-Nluc . Next, the promoter
sequences of Pxx were obtained by PCR-amplified from the
genome of Y. lipolytica using appropriate primers. Finally, the
promoter sequences and linearized pYLXP’-Nluc were assembled to
reporter plasmids pYLXP’-Pxx-Nluc by the Gibson
Assembly method. The constructed plasmids were all sequenced by Sangon
Biotech Co., Ltd (Shanghai, China).
2.3. Yeast transformation by the lithium acetate
method
The standard protocol of the lithium acetate yeast transformation has
been described in the previous report.[3] Briefly,
cells were harvested from 0.5 ml culture solution at 24 h using the YPD
medium by the shaking tube. Then, cells were washed twice using the
phosphate buffer (PBS, 100 mM, pH 7.0) and resuspended by the
transformation solution (105 uL), containing the lithium acetate (2M, 5
uL), PEG4000 (50%, 90 uL), boiled single strand DNA (salmon sperm,
denatured, 5 uL), and reporter plasmids (5 uL). Next, the mixture was
incubated at 39 °C for one hour, which needed to be vortexed for 15
seconds every 15 minutes. Finally, the mixture was spread on the CMS-leu
selected plates. YPD medium used in this study included glucose 20 g/L,
peptone 20 g/L, and yeast extract 10 g/L.
2.4. Shaking flask
cultivations
For this, seed culture was carried out in the seed culture medium (2 mL)
at 30 oC and 250 r.p.m for 48 h, using the shaking
tube. Then, seed culture (0.8 mL) was inoculated into the CSM medium
(C/N=80, 30 mL) in the 250 mL flask and grown at 30 oC
and 250 r.p.m for 120 h. During the process of fermentation, 1 ml
culture solution was sampled every 12 h for luciferase and
OD600 measurements. The seed culture medium contains
yeast nitrogen base without ammonium sulfate (YNB) 1.7 g/L, glucose 20.0
g/L, (NH4)2SO4 5.0 g/L,
and CSM-Leu 0.74 g/L. Moreover, the CSM medium (C/N=80) contains YNB 1.7
g/L, glucose 40.0 g/L,
(NH4)2SO4 5.0 g/L, and
CSM-Leu 0.74 g/L.
2.5. Quantification of cell densities and the promoter
strength
Cell densities of Y. lipolytica were monitored by measuring the
optical density at 600 nm (OD600). The promoter strength
was determined by performing the luciferase whole-cell assay analysis.
In detail, 0.5 ml culture solution was centrifuged at 8,000 r.p.m for 3
min to collect the cell pellet. Then, the collected cell pellets were
washed twice using the phosphate buffer (PBS, 100 mM, pH 7.0), and
resuspended by the same buffer (1 ml). It should be noted that
OD600 of cell pellet suspension solution needed to be
measured and recorded. Next, the reaction mixture of the luciferase
whole-cell assay was prepared for the luciferase activity assay by the
microplate system (following the protocol of Nano-Glo® Luciferase Assay
System kit), which contained the luciferase buffer 100 ul, substrate
2ul, cell pellet suspension solution 10 ul, and sterile water 88 ul. As
a result, the promoter strength was obtained by dividing the luciferase
activity data by the recorded OD600 of the cell pellet
suspension solution.