Strains and media
Yeast strains used in this study were described in Table.2. The astaxanthin producing strain yQDD001 (MATa, His3Δ0, Leu2Δ1, met15Δ0, Ura3Δ0, HO::tR(ccu)J, lys::NAT) was subjected to improve the host's compatibility with crtZ and crtW from different sources by in vitro and in vivo recombination. Selective medium for rearrangement strains were SC-Leu-Ura+G418(synthetic complete medium lacking leucine and uracil with 20 g L−1 glucose and 100ug mL-1 G418), SC-Leu-His+G418 (synthetic complete medium lacking leucine and histidine with 20 g L−1 glucose and 100ug mL-1 G418) and SC-Leu-Ura-His+G418 (synthetic complete medium lacking leucine, uracil, and histidine with 20 g L−1 glucose ). All yeast solid media were added with 20 g L−1 agar. Escherichia coli DH5α purchased from BEIJING Biomed Co., Ltd was used for plasmid transformation. Escherichia coli were cultivated at 37℃ in LB medium (with 10 g L−1 tryptone, 5 g L−1 yeast extract, 10 g L−1 NaCl and 100 ug mL−1 ampicillin). LB solid medium was added with 15 g L−1 agar.
Yeast transformation and assembly
The protocol used for yeast transformation is the LiAc/SS carrier method. Yeast colonies were inoculated into 5 ml of SC-Leu+G418 and grown overnight at 30℃. Then 200ul yeast solution was inoculated into 5 ml of new SC-Leu+G418 cultures. 5-6h after, cultures were washed out twice with ddH2O (double-distilled water) and resuspended in 0.1 M LiAc put on ice until needed. Yeast transformation system contained 620 ul of 50% polyethylene glycol (PEG) with molecular weight 3350, 40ul salmon sperm DNA (SSDNA,100 mg ml−1), 90 ul of 1M LiAc solution. Then, 50ul in vitro or in vivo recombination system was mixed with 100 ul resuspended cells. And the mixed pool was added into LiAc/SS carrier DNA/PEG mixture and stir spirally. Samples were first incubated at 30℃ for 30 min. Then heat-shocked 18min at 42℃ water-bath. 90 ul DMSO was added followed by heat-shocked. Centrifuged and resuspended cells with 400ul 5 mM CaCl2, plated on selective medium after 10min. After culturing for 72 h at 30℃incubator, darker red yeast colonies were selected on synthetic medium.
In vitro recombination
As shown in the additional Fig.2, the donor fragments and acceptors were cut from the plasmids by the enzyme. The 50 ul reaction system of in vitro recombination contained 1000 ng acceptor vector, the donor fragments pool of crtZ and crtW from different sources (1000 ng, respectively) and 2 ul of high concentration Cre recombinase (NEB, M0298M). Refer to previous studies of Zhu et al[28],the Cre recombinase reaction was set up as incubated at 37℃ for 4 h. The Cre enzyme was heat-inactivated for 10 min at 70 ℃. Then the reaction pools were transformed to hosts strains yQDD001 for genotype and phenotype testing. SC–Leu-Ura+G418, SC-Leu-His+G418 and SC-Leu-Ura-His+G418 medium were used to select for recombined constructs. To ensure the quality of color screening, all the yeast transformation were diluted 5-fold and repeated three times in this study. There are thousands of colonies generated for screening high producing strains.
In vivo recombination
As shown in additional Fig.5, The fragments of crtZ and crtW were cut from the plasmids by the NotI enzyme. Refer to the system of in vitro recombination, the 50 ul system of crtZ and crtW inserted randomly in genome contained fragments of crtZ and crtW from different sources (1000 ng, respectively). Then the fragments pool was transformed into the hosts yQDD001. SC–Leu-Ura+G418 or SC-Leu-His+G418 and SC-Leu-Ura-His+G418 medium are used to select for recombined constructs. To ensure the quality of color screening, all the yeast transformation were diluted 5-fold and repeated three times in this study. There are thousands of colonies generated for screening high producing strains.
Shake flask cultivation for astaxanthin production
For shake flask culture, recombinant yeast colonies were inoculated into 5 mL SC-Leu+G418, SC-Leu-Ura+G418, SC-Leu-His+G418, or SC-Leu-Ura-His+G418 liquid medium respectively at 250 r.p.m., 30℃ for 24 h. Then the preculture was inoculated into the corresponding fresh SC defective medium (50 mL) with an initial OD600 of 0.2 for further 14 h cultivation (OD600 ≈ 5.0). Then seed culture was transferred into 50 mL fresh YPD-40 medium (40 g L−1 glucose, 20 g L−1 tryptone and 10 g L−1 yeast extract) at an initial OD600 of 0.1 grown for 84 h with the condition of 250 r.p.m.,30℃. Each sample was performed on technical triplicates.
Growth curve assay
The single colony was cultured to saturation in 5 mL YPD medium at 30 °C. The cultures were inoculated into a 250 mL shake flask containing 50 mL of YPD medium with initial OD600 at 0.1, and cultured at 30 °C, 220 rpm. The OD value was measured at appropriate intervals. Growth curves were plotted using Origin software.
Analysis of astaxanthin production by HPLC
1ml of the saturated culture was centrifuged for 2 min at 12000 g. Cells were washed with 1ml ddwater twice and resuspended in 1 ml of 3 M HCl. The resuspended cells were heated in the boiling water bath for 2 min and then cooled in ice-bath for 3 min, repeating three times. Then the samples were washed twice with ddwater to wash out HCl and harvested by centrifugation at 12000g for 2min. After removal of the supernatant, the cells were resuspended in 500ul acetone and vortexed for 20 min. Acetone extracts were centrifuged (13000g, 15min) and filtered with a 0.22 um filter for subsequent. Astaxanthin yield of samples was determined by HPLC (Waters 2695) equipped with HyPURTY C18 column (150mm ×4.6 mm, Thermo Scientific) and UV detection at 450 nm and 470 nm at 25 ℃. The following two buffers were used:A buffer, acetonitrile/Water (9:1 vol/vol) and B buffer, methanol/2-propanol (3:2 vol/vol). The flow rate of the mobile phase was 1 mL/min., and the solvent gradient was as follows: from 0 to 15 min for 100% to 10% of A buffer and 0% to 90% of B buffer, and then from 16 to 30 min for 10% of A buffer and 90% of B buffer,then from 31-35 min for 10% to 100% of A buffer and 90% to 0% of B buffer, at last from 35 to 55 min for 100% of A and 0% of B buffer. Each sample was performed on technical triplicates.
PCRtag analysis
15 ul PCR reaction system contained 7.5 ul 2×rapid Taq master mix (Vazyme), 0.3 uL forward primer (10 uM), 0.3 ul reverse primer (10 uM), 1 uL genome DNA, and 4.9 uL ddH2O. The procedure: 95 ℃/3 min, 30 cycles of (95℃/15s, 53℃/30 s,72 ℃/15 s), and 72 ℃/5 min. Agarose gel electrophoresis was used for PCR analysis. All primers used in this study were listed in additional Table.2.
Screening and verification of selected strains
For preliminary screening, darker red and big colonies were selected on selective media. Candidate strains were verified on SD media (synthetic complete medium with 20 g L−1 glucose) using a 10-fold serial dilution assay.
Extraction of the yeast genomic DNA
Strains were cultured overnight to saturation. Centrifuged at 12, 000 rpm to harvest cells. 200 μl breaking buffer (500 mM.L-1 NaCl, 200 mM.L-1 Tris-HCl, 100 mM.L-1 EDTA, 1% SDS), 200 μl silica sand and 200 μl phenol/chloroform/isoamyl alcohol (25:24:1) were added to cells tube. Disrupted cells by the vortex mixer for 20 minutes. Then added 1 mL cold ethanol to the supernatant, mixed and centrifuged at 4 ℃ for 10 minutes. The precipitate was washed with 75% cold ethanol and dried at 37 ℃. 200 μl ddH2O was added to dissolve the yeast genome DNA. Stored the genome DNA at -20 ℃.
Quantitative real-time PCR (qPCR) analysis
qPCR was applied to quantify copy numbers of the gene in engineered strains. The template in the qPCR analysis was yeast genomic DNA. Reference primers were selected from gene ALG9. and the target primers were selected from the cassettes of crtZ and crtW respectively. Strain with a single copy of crtZ and crtW was used as the reference strain. The copy numbers were determined by comparing the Ct values of crtZ or crtW and the reference gene ALG9 using the 2−ΔΔC t method. The relative ratio of crtZ or crtW was calculated as 2−Δt(crtZ) or 2−ΔCt(crtW). Unique Aptamer TM qPCR SYBR Green Master Mix (Beijing Novogene Bioinformatics Technology Co., Ltd) was used for the qPCR reaction, and the equipment was Quantagene q225 (Novogene). The reaction procedure was performed as follows: precycling,95℃/300 s, 40 cycles of (95℃/10 s, 57℃/20 s, 72℃/20 s), melt curve, which started from 60℃to 95℃.