I. Preparation of media

1. LB medium:

1) Weigh 10 g tryptone, 5 g yeast extract and 10 g NaCl using an electronic balance, then add these components to an Erlenmeyer flask.

2) Use a graduated cylinder to measure 1 L ddH₂O, and then add water into the Erlenmeyer flask mentioned above.

3) Dissolve the components by shaking the flask.

4) Separate the solution equally, label one as LB liqud and the other as LB solid.

5) Add 7.5 g agar into the LB solid medium.

6) Sterilize by autoclaving at 121 ℃ for 20 min.

7) Allow LB solid medium to cool to 50-60 °C after autoclaving.

8) Add antibiotics to the solid LB medium, mix well and pour the plates in a laminar flow hood. Make it solidify.

9) Store prepared LB agar plates at 4 °C.

2. YPD medium:

1) Weigh 20 g glucose and measure 500 mL ddH₂O, add in an Erlenmeyer flask labeled glucose.

2) Shake the flask to dissolve the glucose.

3) Weigh 20 g tryptone and 10 g yeast extract using an electronic balance, measure 500 mL ddH₂O, and add to another Erlenmeyer flask.

4) Shake the Erlenmeyer flask evenly.

5) Separate the solution equally, label one as YPD liquid and the other as YPD solid.

6) Add 7.5 g agar into the YPD solid medium.

7) Sterilize by autoclaving at 121 ℃ for 20 min.

8) Allow YPD solid medium to cool to 50-60 °C after autoclaving.

9) Add antibiotics to the solid YPD medium, mix well and pour the plates in a laminar flow hood. Make it solidify.

10) Store prepared YPD agar plates at 4 °C.

 

II. Polymerase Chain Reaction (PCR)

A. AppA:

1) Use the E. coli DH5α genomic DNA as template.

2) Use AppA-F and AppA-R as forward and reverse primers, respectively.

B. An_phy33:

1) Use the synthesized plasimd containing An_phy33 gene as the template DNA.

2) Use An_phy33-F and An_phy33-R as forward and reverse primers, respectively.

The reaction system for PCR amplification are shown in the table below:

 

The reaction procedure for PCR amplification are shown in the table below:

Notice: Repeat step 2 to step 4 for 28 times.

 

III. Inoculation of E. coli and yeast

1. Inoculation of E. coli:

1) Measure 3 mL LB liquid medium, add 3 μL ampicillin to a final concentration of 50 μg/mL, and inoculate the E. coli strain.

2) Incubate the E. coli culture in a 37 ℃ shaker at 220 rpm for 12-16 h.

1. Inoculation of yeast:

1) Measure 4 mL YPD liquid medium, add 4 μL ampicillin to a final concentration of 50 μg/mL, and inoculate the BY4741 yeast strain.

2) Incubate the yeast culture in a 37 ℃ shaker at 220 rpm for 24-48 h.

 

IV. Agarose gel electrophoresis

1. Preparation of agarose gel

1) Add 100 mL 1×TAE buffer and 1 g agarose to an Erlenmeyer flask, and place it in a microwave oven on high for 2 min.

2) Take it out after the solution turns transparent and shake it evenly.

3) Add 2 μL nucleic acid dye GelRed 10000 ×to the molten agarose.

4) Assemble gel tray and insert comb into mold.

5) Pour the liquid agarose into the mold to a depth of 3-4 mm, avoiding air bubbles.

6) Wait for solidification of the gel at room temperature.

7) Carefully remove comb and transfer solidified gel to electrophoresis tank.

8) Completely submerge gel in 1 ×TAE buffer.

2. Loading samples

1) Add DNA loading buffer into samples to dye them blue, then load into wells sequentially. Avoid contacting gel when pipetting.

2) Add 10 μL of Trans15K DNA Marker into a lane.

3. Electrophoresis

1) Place wells at the negative electrode, and cover tank.

2) Turn on the power, and run the agarose gel at 180V for 20 min.

4. Visualizing DNA bands

1) Turn off power, and remove gel.

2) Place the gel under the Ultra Slim LED Illuminator to visualize the results.

3) Take out the gel, and put it under gel-imaging system to save data.

 

V. Gel Extraction (with DNA Gel Extraction Kit)

1. Preparation

1) Check whether anhydrous ethanol has been added to the Wash Solution.

2) Make sure Buffer B2 does not appear to be precipitated.

3) Set the constant temperature water bath to 50 ℃.

2. Extraction

1) Rapidly excise the DNA band of interest under UV light using a clean scalpel.

2) Transfer the gel slices into centrifuge tubes, and measure the weight of the gel slices.

3) Add 3 times of sample volume of Buffer B2 into the centrifuge tubes, and mix until gel completely dissolved.

4) Transfer the solution into the absorption column, and centrifuge at 8000 ×g for 30 sec. Pour off the filtrate in the collection tube.

5) Add 500 μL of Wash Solution and centrifuge at 9000 ×g for 30 sec. Pour off the filtrate in the collection tube.

6) Repeat wash step.

7) Centrifuge the empty column at 9000 ×g for 1 min.

8) Place the absorption column into a clean 1.5 mL centrifuge tube, add 15-40 μL Elution Buffer to the center of the absorption membrane, and centrifuge at 9000 ×g for 1 min. Store the DNA solution in the tube.

 

VI. Plasmid extraction (Plasmid Mini-PREPS Kit)

1. Preparation

1) Make sure RNase A has been added to Buffer SP1, anhydrous ethanol has been added to Wash Solution, and no precipitation appears in Buffer SP2 and SP3.

2. Equilibrate the adsorption column

1) Add 500 μL of Buffer S to the adsorption column and place a collection tube below it.

2) Centrifuge at 12,000 ×g for 1 min, discard the waste liquid in the collection tube and put the adsorption column back into the collection tube.

3. Transfer all the E. coli culture into a 2 mL centrifuge tube, centrifuge at 12,000 ×g for 2 min to collect the bacteria. Discard the supernatant and retain the precipitate.

4. Add 250 μL of Buffer SP1 to the 2 mL centrifuge tube, and thoroughly suspend the precipitate with pipette.

5. Add 250 μL of Buffer SP2, and gently invert the tube 5-10 times to mix the solution.

6. Add 350 μL of Buffer SP3 and mix the solution by gently inverting the tube 5-10 times.

7. Centrifuge at 12,000 ×g for 5-10 min. Transfer 700 μL of the supernatant to the adsorption column, centrifuge at 8,000 ×g for 30 sec, and discard the filtrate in the collection tube.

8. Add 500 μL of Wash Solution, centrifuge at 9,000 ×g for 30 sec, and discard the filtrate in the collection tube.

9. Repeat wash step.

10. Centrifuge the empty column at 9,000 ×g for 1 min.

11. Place the column into a clean 1.5 mL centrifuge tube, add 25 μL of Elution Buffer to the center of the adsorption membrane. Incubate for 1 min at room temperature, centrifuge at 9,000 ×g for 1 min, and keep the DNA solution in the tube.

 

VII. Double enzyme digestion of fragments and vectors

1. The double digestion system for PCR products is shown in the table below:

Reagent	Volume (μL)
PCR products (AppA / An_phy33)	20
SphⅠ	1
AvrⅡ	1
Buffer (CutSmart)	5
ddH2O	23
Total	50

 

2. The double digestion system for vector is shown in the table below:

Reagent	Volume (μL)
plasmid (pYES2-Hyg-GAL1p-α-SED1-RPL41Bt)	30
SphⅠ	1
AvrⅡ	1
Buffer (CutSmart)	5
ddH2O	13
Total	50

The enzyme digestion systems are incubated at 37 ℃ for 3 h.

 

VIII. Ligation of recombinant plasmids

1. Plasmid 1: pYES2-Hyg-GAL1p-α-AppA-SED1-RPL41Bt

Reagent	Volume (μL）
plasmid (pYES2-Hyg-GAL1p-α-SED1-RPL41Bt)	13.6
AppA	3.4
T4 ligase	1
T4 ligase buffer (10×)	2
Total	20

 

2. Plasmid 2: pYES2-Hyg-GAL1p-α-An_phy33-SED1-RPL41Bt

Reagent	Volume (μL）
plasmid (pYES2-Hyg-GAL1p-α-SED1-RPL41Bt)	15
An_ phy33	2
T4 ligase	1
T4 ligase buffer (10×)	2
Total	20

The ligation systems are incubated at 16 ℃ for 16 h, followed by 5 min at 65 ℃ to inactivate the ligase.

 

IX. Transformation of recombinant plasmids to E. coli DH5α

1. Add 5 μL of pYES2-Hyg-GAL1p-α-AppA-SED1-RPL41Bt plasmid and pYES2-Hyg-GAL1p-α-An_phy33-SED1-RPL41Bt plasmid into 50 μL of E. coli DH5α competent cells, respectively. Incubate the mixture on ice for 30 min.

2. Heat shock at 42 °C for 90 sec, and put the mixture to the ice for 4-5 min.

3. Add the LB culture into the tubes to 1 mL, and shake the tubes at 37 ℃, 220 rpm for 30-60 min.

4. Centrifuge it at 9000 ×g for 1 min, remove 800 μL of supernatant, and resuspend the cells in the remaining LB culture.

5. Spread 50 μL cells onto LB agar plates containing ampicillin and incubate overnight at 37 ℃.

 

X. PCR identification of transformants

1. Place 50 μL of Lysis Buffer for Microorganism to Direct PCR in a sterilized microtube. Pick a single colony with a sterilized toothpick or lance tip, place it in the microtube and stir a few times before removing it. (Note: Do not leave the toothpick in the microtube for too long to prevent affecting the volume of lysate and PCR amplification.)

2. After heat denaturation at 80 °C for 15 min, centrifuge at low speed and take 1~5 μL of supernatant after lysis as PCR template.

The PCR reaction system is shown in the table below:

Reagent	Volume (μL)
Lysis supernatant	1
Premix Taq (Ex Taq Version 2.0)	10
Forward Primer (10 μM  )	1
Reverse Primer (10 μM)	1
sterilized water	7

 

The PCR reaction condition is shown in the table below:

Step	Temperature (˚C)	Time (sec)
1	94	600
2	94	30
3	55	30
4	72	120
5	72	300
6	4	forever

Notice: Repeat step 2 to step 4 for 28 times.

 

3. Take positive transformants and add 3 mL LB culture medium, add 3 μL ampicillin, shake bacteria, and incubate at 37 ℃ at 220 rpm for 12-16 h

4. Inoculate the positive transformants in 3 mL LB culture medium containing 50 μg/mL ampicillin at a 37 ℃ shaker with 220 rpm for 12-16 h.

5. Extraction the plasmid from overnight cultures for yeast transformation.

 

XI. Preparation of Saccharomyces cerevisiae Competent Cell

1. Select single bacterial colony from YPD plate, and inoculate it into YPD liquid medium for 12-24 h.

2. Measure the OD₆₀₀ value of the overnight culture (between 4.0 and 5.0). Notably, a 10-20 dilution of the culture solution is required for the measurement.

3. Dilute the overnight culture to OD₆₀₀ value of 0.2~0.4, and then transfer into shaker.

4. The diluted culture is continued to incubate in a shaker at 30 ℃, 220 rpm for 4-5 h, until OD₆₀₀ reaches 0.8-1.0.

5. Centrifuge the culture at 3000 ×g at 20 ℃ for 5 min, and discard the supernatant.

6. Resuspend the pellet in ddH₂O, and centrifuge at 3000 ×g at 20 ℃ for 5 min. Discard the supernatant.

7. Repeat water wash step.

8. Resuspend the pellet in 1 mL ddH₂O into 1.5 mL centrifuge tube. Centrifuge at 12000 ×g for 30 sec for 5 min, and discard the supernatant.

9. Resuspend the pellet in 1mL Cell Freezing Medium. Aliquot 100 μL per tube, and freeze at -80°C.

 

XII. Plasmid Transformation to Saccharomyces cerevisiae BY4741

1. Place the plasmid in a 95 ℃ water bath for 10 min, and then transfer it onto ice.

2. Take out 100 μL yeast competent cells and place them in a 37 ℃ water bath for 20 sec. Centrifuge it at 12000 ×g for 30 sec and remove the supernatant.

3. Add 36 μL of 1.0 M LiAc, 50 μL linearized plasmid DNA, and 260 μL PEG 3350 into the tube with yeast competent cell precipitate. Mix it thoroughly with pipette.

4. Heat shock the cells at 42 °C for 25 min.

5. Centrifuge at 12000 ×g for 30 sec, remove the supernatant and place it on ice for 5 min.

6. Resuspend the pellet in 1 mL ddH₂O.

7. Spread 100 μL of the mixture on the YPD culture containing hygromycin (100 μg/mL), and incubate at 30 ℃ for 48 h.

 

XIII. PCR identification of positive transformants 

1. Place 50 μL of Lysis Buffer for Microorganism to Direct PCR in a sterilized microtube. Pick a single colony with a sterilized toothpick or lance tip, place it in the microtube, and stir a few times before removing it. (Note: Do not leave the toothpick in the microtube for too long to prevent affecting the volume of lysate and PCR amplification.)

2. After heat denaturation at 80 °C for 15 min, centrifuge at low speed and take 1~5 μL of supernatant after lysis as a template for PCR reaction.

The PCR reaction system is shown in the table below:

Reagent	Volume (μL)
Lysis supernatant	1
Premix Taq (Ex Taq Version 2.0)	10
Forward Primer (10 μM)	1
Reverse Primer (10 μM)	1
sterilized water	7

 

The PCR reaction condition is shown in the table below:

Step	Temperature (˚C)	Time (sec)
1	94	600
2	94	30
3	55	30
4	72	120
5	72	300
6	4	forever

Notice: Repeat step 2 to step 4 for 28 times.

 

XIV. Protein expression

1. Take a positive transformant and add 3 mL YPD culture medium containing 100 μg/mL hygromycin. Grow at 30°C, 240 rpm to OD₆₀₀ 0.4-0.6 (∼24 h).

2. Centrifuge the yeast culture and remove the supernatant.

3. Resuspend the pellet with sterile water to remove glucose and transfer it to a 50 mL YPD culture medium containing 100 μg/mL hygromycin.

4. Add galactose to induce promoter and express surface-displayed phytase for 24-48 h.

 

XV. Preparation of samples

1. Preparation of yeast surface-displayed phytase

a. Collect yeast by centrifugation at 8000 rpm for 5 min and measure the wet weight.

b. Washing the yeast cells twice with 50 mM sodium acetate buffer (pH 5.0). Retain for the determination of surface phytase activity.

2. Preparation of yeast phytase from the yeast cell wall

a. Collect yeast by centrifugation at 8000 rpm for 5 min and measure the wet weight.

b. Washing the yeast cells twice with 50 mM sodium acetate buffer (pH 5.0).

c. Sonicate cells at 30% power for 5 min at 0°C, and the lysate was centrifuged at 4 °C, 8000 rpm for 5 min.

d. Washed the lysate precipitation twice with 50 mM sodium acetate buffer (pH 5.0). Retain for the determination of cell wall phytase activity.

 

XVI. Phytase activity

1. Prepare substrate solution (500 mM sodium phytate, 50 mM acetic acid/sodium acetate buffer, pH 5.0) and termination reaction solution (15% (w/v) trichloroacetic acid).

2. Mix 200 μL phytase-displayed yeast with 200 μL substrate solution. Incubate 30 min at 37 °C.

3. Terminate the reaction by adding 100 μL of the termination reaction solution and measure the absorbance value at 700 nm spectrophotometrically.

4. Generate standard curve using K₂HPO₄ standards from 0-1.0 mg/mL. Calculate free phosphate released.

5. A phytase unit activity (U) was defined as the amount of enzyme required to release 1.0 μmol/min of inorganic phosphorus from 5 mmol/L sodium phytate under optimal conditions.

6. Determination of phytase under different pH:

Prepare phytase-displayed yeast cell suspensions with pH ranging from 1-8. Carry out the enzyme activity assay according to the above steps.

7. Determination of phytase under different temperature:

Incubate the phytase-displayed yeast cell suspension and the reaction substrate at different temperatures (30-60 ℃). Carry out the enzyme activity measurement according to the above steps.