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.