2. Material and methods
2.1 Gene amplification and plasmid construction
Escherichia coli DH5α was employed for the replication and
construction of plasmids. The information on all yeast strains and
plasmids utilized in this research are presented in Table 1 and Table
S1. All genes utilized in this research presented in Table S2, includingHaTAL (from Hemiphyllodactylus aurantiacus ), MtPDH1(from Medicago truncatula ), OsNOMT (from Oryza
sativa ), PhCHS (from Petunia hybrida ), and MsCHI(from Medicago sativa ). All primers used in this study have been
listed in Table S3. The plasmids of pH1, pH2, pH3, pH4, pH5, and pH6
(Lian & Zhao, 2015) were utilized as empty vectors for PCR
amplification of all native promoters, genes, and terminators inS. cerevisiae CEN.PK2-1C. Codon-optimized heterologous genes were
amplified by PCR using synthetic fragments or available plasmids. Genes
of AtPAL2 , AtC4H, AtATR2, and At4CL1 were amplified
from plasmids of pH1-AtPAL2, pH2-AtC4H,
pCfB2767, and
pCfB2584, respectively.EcaroL and HaTAL were amplified from E. coligenomic DNA and plasmid of pTAL. The mutant genes ofARO4K229L , ARO7G141S ,
and ACC1 S659A, S1157A were obtained by overlap
extension PCR.
These candidate genes, promoters, or terminators were then cloned into
template plasmids (pH1, pH2, pH3, pH4, pH5, pH6)
(Lian & Zhao, 2015), using
restricted ligation or Gibson assembly to produce gene cassette
plasmids. In addition, these gene cassettes are amplified and assembled
by using DNA assembly methods into multi-gene pathways. Then they were
integrated into genomic locis where heterologous genes could be
efficiently and stably expressed (Apel et al., 2017). Using plasmid
pKan10-ADE2.1 (Lian et al., 2018) as a template, all guide RNA (gRNA)
plasmids were constructed via Gibson assembly with the corresponding
primers. Guide RNAs and integration sites were designed on E-CRISP
website (http://www.e‐crisp.org ) (Heigwer et al., 2014) and
listed in Table S4.
2.3 Strain construction and cultivation conditions
All strains used or constructed were listed in Table 1, gene knockout
and insertion of DNA fragments in Saccharomyces cerevisiae were
operated by CRISPR/Cas9 system (Stovicek et al., 2015). YT00
(CEN.PK2-1C, IX1 :: TEFp-SpCas9-ADH2t ) (Xiao et al., 2022)
was adopted as the host strain for the integration of sakuranetin
synthesis pathway genes. LiAc/ssDNA/PEG method was used to co-transform
an equal amount of purified linearized fragments (50-100 ng/kb) with the
corresponding gRNA plasmids (~300-500 ng) into S.
cerevisiae , and YPD agar plate containing 200 μg/mL G418 was used for
the selection of the resulting strains. The clones were selected to
verify whether knockout or integrate into the corresponding position of
genome by PCR. And then, the right module-integrated clones were
cultured in YPD for an entire night before being streaked onto plates
without antibiotics to loop out of gRNA vectors.
E. coli was cultivated at 37℃ in Luria-Bertani medium containing
10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 100 μg/mL of
ampicillin or kanamycin if necessary. S. cerevisiae was
cultivated in YPD medium containing 20 g/L peptones, 10 g/L yeast
extract, 20 g/L glucose, and 200 μg/mL of G418 for the selection of
engineered strains. For shaking-flasks fermentation, the engineered
yeast strains were selected to cultivate in 5 mL YPD tubes under 30°C
and 220 rpm. The broth was then inoculated into 50 mL of basic medium
with an initial OD660 of 0.05, and incubated under 30°C
and 220 rpm.
For bioreactor cultivation, the selected yeast strain was incubated into
a 5 mL YPD tube at 30℃, 220 rpm for 24h, 4% of seed solution was then
transferred to 250 mL flasks, which contains 50 mL of YPD medium, and
cultivated for 22 h. The obtained culture was transferred to a 1-L
bioreactor containing 500 mL of YPD medium (2%) with an initial
OD660 of 0.68. The fermentation was carried out in a
bioreactor at 30 C, 400 rpm, with the pH maintained at 5.5, and the
airflow rate was 1 L/min by automatic addition of control. The
concentration of sakuranetin and OD660 was determined by
regular sampling during fermentation.
2.4 Analysis of the metabolites
To determine the concentration of p -coumaric acid, naringenin,
and sakuranetin, 800 μL ethyl acetate was added to 800 μL S.
cerevisiae culture. After mixing by vortex and centrifugation at 12,000
rpm for 10 min, the top layer of 400 μL (ethyl acetate organic phase)
was transferred to a 2 mL centrifuge tube. Then 400 μL of ethyl acetate
was added to the remaining S. cerevisiae culture for the second
extraction. A total of 800 μL ethyl acetate organic phase extracts were
mixed and concentrated in a vacuum centrifuge at 50 °C for 40 min. Then
the dry residue was dissolved in 800 μl methanol. Before HPLC analysis,
the samples were filtered via a 0.22 μm organic filter membrane.
HPLC analysis was performed using Agilent 1260 HPLC system and a C18
column (250×4.6 mm, 5 μm). The mobile phases consisted of solvent A
(0.2% acetic acid in water) and B (0.2% acetic acid in methanol). The
gradient program was performed as follows: 0.00-10.00 min:75-25%
B;10.00-20.00 min:25% B; 20.00-23.00 min:25%-75% B;23.00-25.00
min: 75% B. The flow rate was 1mL/min and the injection volume was 10
μL with stable column temperature at 30℃, p -coumaric acid,
naringenin, and sakuranetin were detected at 8.2 min (308 nm), 11.5 min
(288 nm), and 13.9 min (288 nm), respectively. Thermo Ultimate 3000 UPLC
equipped with a mass spectrometer (LC-MS) was used to analyze the mass
values of sakuranetin. The mass spectra data were processed and analyzed
by using the software UniDec (Marty et al., 2015) to produce meaningful
mass distributions. The optical density at 660 nm was measured by an
ultraviolet spectrophotometer to monitor cell concentration. A biosensor
analyzer SBA-40D (Shandong, China ) was utilized to analyze the
concentration of glucose in the supernatant.