ON THIS PAGE

Experiment

Plasmid design and chemical synthesis

For astaxanthin biosynthesis in E.coli, seven key genes including HpCrtE, PanCrtB, PanCrtI, PagCrtY, idi, HpCrtZ, BreCrtW, were chemically synthesized by GenScript Biotech Corporation (Nanjing, China) and inserted into the commonly used bacteria vector pET-Duet-1 for expression as following design (Fig. 1).

Briefly, CDs of HpCrtE, PanCrtI, and PanCrtB were sequentially followed by the first T7 promoter while CDs of PagCrtY, idi, HpCrtZ, BreCrtW were sequentially followed by the second T7 promoter. The 14 bp nucleotide was used to space each gene. The synthesized plasmid was delivered into E.coli Top10 cells for storage.

Figure 1. The schematic map of plasmid used for astaxanthin production in E. coli .

Plasmid DNA extraction

Plasmid was extracted from E. coli cells using Omega Plasmid Mini Kit. Detailed procedures are as following:

  1. Transfer overnight bacterial cultures to a 2.0 mL Eppendorf tubes and centrifuged at 12,000 rpm for 1 minute and discard the supernatant
  2. Add 250 µL of Solution I (contains RNase) cells to the cell pellet and vertex to lyse cells
  3. Add 250 µL of Solution II into the mixture and gently mixed
  4. Add 350 µL Solution III to the mixture and invert the tube to form the flocculent material
  5. Centrifuge at 13,000 ×g for 10 minutes
  6. Transfer the supernatant to the spin column
  7. Centrifuge at 13,000 ×g for 1 minutes and discard the liquid in the collection tube
  8. Add 500 µL HBC Buffer to the spin column, centrifuge for 1 minute, and discard the liquid from the collection tube
  9. Add 750 µL DNA Wash Buffer to the spin column, centrifuge at 13,000 ×g for 1 minute, and discard the liquid from the collection tube
  10. Repeat the previous step
  11. Without adding any reagents to the spin column, centrifuge at 13,000 ×g for 2 minutes, and discard the liquid from the collection tube
  12. Stand for 5 minutes to allow residual ethanol to evaporate completely from the spin column
  13. Transfer the spin column to a new 1.5 mL Eppendorf tube, add 50 µL ddH₂O, and stand at room temperature for 2 minutes
  14. Centrifuge at 13,000 ×g for 2 minutes to collect the plasmid DNA solution

The obtained plasmid DNA was quantified by NanoDrop 2000 and stored at -20°C for future use.

Restriction enzyme digestion

Prepare the reaction in a 0.2 mL EP tube on ice as shown in Table 1. After adding all agents, briefly centrifuge the mixture and then incubate at 37°C for 1 hour.

Table 1. The agent and corresponding amount in a digestion reaction.
Agent Amount
Plasmid DNA 1 µg
FastDigest Pac I 1 µL
FastDigest Nde I 1 µL
10 × FastDigest Green Buffer 2 µL
ddH₂O To final 20 µL

Agarose gel electrophoresis

To prepare 1% agarose gel, detailed procedures are as following:

  1. Accurately weigh 0.5 g agarose into a 150 mL conical flask and add 50 mL 1× TAE electrophoresis buffer
  2. Place the conical flask in a microwave oven and heat for 1-2 minutes until the agarose was completely dissolved
  3. After cooling down to 60-70°C, add Goldview dye into the gel solution to achieve a final concentration of 0.5 μg/mL
  4. Gently swirl to mix the gel solution
  5. Position the comb within the gel casting tray
  6. Slowly pour the mixture into the tray, taking care to minimize bubble formation
  7. Allow 30 minutes for gel solidification
  8. Gently remove the comb from the vertical position
  9. Transfer the entire casting tray into the horizontal electrophoresis chamber containing enough 1× TAE buffer
  10. Load proper amount of samples into the gel well
  11. Set up the voltage at 120 V for electrophoresis
  12. After 20 minutes, record the gel using BioRad ChemiDoc XRS+ Gel Imaging System

Transformation of E. coli

E. coli BL21(DE3) was used as the factory for pigment production. Competent cells of BL21(DE3) were purchased from Weidi Biotech (Weidi Biotechnology Co., Ltd, Shanghai, China). Heat-shock method was used for E. coli transformation. Detailed procedures are as following:

  1. Thaw frozen BL21(DE3) competent cells slowly on ice for 5 minutes
  2. Add 50 ng plasmid DNA to 100 µL competent cells
  3. Gently mix by tapping the bottle of tubes
  4. Incubate the tube on ice for 30 minutes
  5. Promptly transfer the tube to a pre-warmed 42°C water bath for 45 seconds
  6. Immediately return tube to the ice bath and allow to stand for 2 minutes
  7. Add 700 µL LB liquid medium to the tube
  8. Recover the cells by incubation at 37°C with shaking at 200 rpm in a shaking incubator
  9. Take 100 µL of the recovered culture and spread it evenly onto LB solid plates containing 100 µg/mL ampicillin
  10. Incubate the plates upside down in a 37°C incubator for 12-16 hours until single colonies appear

To prepare 1 L LB medium, 10 g of sodium chloride, 10 g of peptone, and 5 g of yeast extract were completely dissolved in dH₂O to final volume of 1 L. For solid medium preparation, additional 15 g of agar powder was added. The prepared medium was autoclaved at 121°C for 20 minutes.

Single colony selection and seeding culture

Detailed procedures for seedling culture are as following:

  1. Pick up single colony with red color by a sterilized toothpick in a laminar flow hood
  2. Put the toothpick into the 15 mL culture tube containing 3 mL LB medium supplied with 100 mg/ml ampicillin
  3. To grow the seed cells, the culture tube was put into a 37°C incubator with shaking at 220 rpm for 12-16 hours

E. coli culture and IPTG induction

In a laminar flow hood, 1 mL seed culture was inoculated into a 250 mL flask containing 100 mL LB medium supplied with 100 mg/ml ampicillin. The flask was put into a shaker at 30°C with 220 rpm for about 2 hours until OD₆₀₀ reaches 0.5-0.6. IPTG at a final concentration of 0.1 mM was added into the culture and cells were grown for another 5 hours.

Cells were harvested using 50 mL tube by centrifugation at 8000 rpm for 8 minutes at 4°C. To remove the residual culture medium, cells were washed by sterilized ddH₂O and harvested again by centrifugation at 8000 rpm for 8 minutes at 4°C[1].

Culturing E. coli in large scal and IPTG induction

Firstly, picked single colony containing the astaxanthin synthesis plasmid from the plates and cultured in 250 mL Erlenmeyer flasks containing 80 mL LB medium (100 μg/mL ampicillin). In a laminar flow hood, 80 mL seed culture was inoculated into a 1000 mL flask containing 800 mL LB medium supplied with 100 μg/mL ampicillin. The flask was put into a shaker at 30°C with 220 rpm until OD₆₀₀ reaches 0.6. IPTG at a final concentration of 0.1 mM was added into the culture and cells were grown for another 5 hours. Cells were harvested by centrifugation at 8000 rpm for 8 minutes at 4°C. To remove the residual culture medium, cells were washed by sterilized ddH₂O and harvested again by centrifugation at 8000 rpm for 8 minutes at 4°C[1].

Pigment extraction from E. coli cells

Harvested cells were dried in a vacuum freeze dryer. Firstly, seal the centrifuge tube with cell pellet with parafilm and punch 5-10 small holes with toothpick. Secondly, freeze the cell pellet by liquid nitrogen. Lastly, place the frozen samples in a rack upstraight and put the rack into the cold trap chamber. After 48 hours, cell pellets were dried and subjected to pigment extraction.

Detailed procedures for pigment extraction are as following:

  1. Crushed the dried cell pellet into fine powder
  2. Exactly weigh 10 mg powder into a 2.0 mL centrifuge tube
  3. Add 50 mL glass beads (0.5 mm in diameter) and 1 mL of methanol:isopropanol (8:2) solution into the tube
  4. Use a low-temperature grinder (setting parameters including temperature at 4°C, frequency at 60Hz, cycle at 15-second bursts with 5-second pauses) to break down the cells for 10 minutes
  5. Centrifuge at 12,000 rpm for 2 minutes at 4°C
  6. Suck the supernatant with a syringe and filter through a 0.22 µm Nylon filter to a 1.5 mL brown centrifuge tube[1-2].

Astaxanthin quantification by HPLC

The astaxanthin content in the pigment extracts from E. coli cells was determined by HPLC. Shimadzu LC-2030C 3D system (Shimadzu, Kyoto, Japan) HPLC instrument coupled with Phenomenex Gemini-NX C18 column (5 µm, 150 × 3.0 mm, Phenomenex Inc., Aschaffenburg, Germany) was used.

The mobile phases include ultrapure water (Solution A) and methanol: isopropanol (8:2) (Solution B). The flow rate is 0.6 mL/min, injection volume was 10 µL, column temperature was 35 °C, and DAD detection at 450 and 475 nm.

The gradient for HPLC analysis was initial conditions at 15% A, 85% B; 0-10 min from initial condition to 100% B; 10-12 min maintained at 100% B; 12-12.1 min back to initial conditions; and 12.1-18 min recalibrate the column with initial condition. Chromatograms and the peak area of astaxanthin was recorded using LabSolution software.

Calibration curve was used to calculate the astaxanthin concentration in the detected sample. Firstly, commercial astaxanthin standards were prepared at the concentrations of 1, 0.5, 0.25, and 0.1 µg/mL. Subsequently, standards were run by HPLC. Finally, the concentrations of standards (y-axis) were plotted with corresponding peak areas (x-axis) to construct the standard curve.

The formula used for astaxanthin content calculation was:

Astaxanthin content in µg/mg DCW (dried cell weight) = asta (conc. µg/mL) × 1 (mL) / W (mg)

Where asta (conc. µg/mL) is the calculated astaxanthin concentration in the detected sample using peak area based on the standard curve and W is the exact weight of cells subjected for pigment extraction[2].

References

[1] Ma T, Zhou Y, Li X, Zhu F, Cheng Y, Liu Y, Deng Z, Liu T. Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp. ATCC 55669 for heterologous overproduction in E. coli. Biotechnol J. 2016;11:228–237.

[2] Huang D, Liu W, Li A, Wang C, Hu Z. Discovery of geranylgeranyl pyrophosphate synthase (GGPPS) paralogs from Haematococcus pluvialis based on Iso-Seq analysis and their function on astaxanthin biosynthesis. Mar Drugs. 2019;17:696.