1.1 Prepare solid LB medium

Main experimental instrument:

Experiment equipment	Experiment reagent
Balance	Tryptone
Weighing paper	Yeast extract
Volumetric flask	NaCl
Cylinder	Agar
Autoclave	DDwater

Process:

1. Turn on the balance
2. Put the weighing paper.
3. Use the balance to measure out 2.5g of Tryptone, 1.25g of Yeast extract, 2.5g of NaCl, and 3.75g of Agar. Place them in a volumetric flask.
4. Measure out 250ml of ddwater using a cylinder.
5. Add the material above together.
6. Align the air hole on the seal with the mouth of the bottle and tie it up with a rubber band.
7. Put it in the Autoclave at 121℃ for 20min
8. Let it cool for 1 hour

1.2 Allocation of antibiotics Amp⁺ and SmR

Main experimental instruments:

Experiment equipment	Experiment reagent
Balance	DDwater
Cylinder	Amp+
Centrifuge tube
Sterilizing table
UV light
Culture tube
Sterilizing filter

Process:

1. Measure out 1g of Amp⁺ using the balance.
2. Measure 10ml of water using the cylinder
3. Add them together into the centrifuge tube
4. Shake them and wait until fully mixed.
5. Put the equipment on the sterilizing table.
6. Turn on the UV light and wait for 10 minutes.
7. Take out a cell culture tube
8. Place a sterilizing filter on it
9. Use the syringe to suck up the Amp⁺ solution
10. transfer it into the cell culture tube through the sterilizing filter
11. Close the lid
12. Seal it with sealing film

1.3 Culture bacteria—PK, EPDH, and PTase

Main experimental instruments:

Experiment equipment	Experiment reagent
Bechtop	LB medium
UV lamp	PK, EPDH, and PTase
Shaker

Process:

1. Put the materials on the sterilizing table.
2. Turn on the UV light and wait for 10 minutes.
3. Take out the culture tube and add in Enzyme PK, Pet-Ptac-T7, PTase, EPDH, and liquid LB using the syringe for 3ml.
4. Add 5 μL of any of the solutions into the culture tube
5. Seal the lid of the culture tube with the sealing film.
6. Exchange the head of the pipette.
7. Repeat the pattern until all bacteria have been used
8. Put all into the shaker at 37℃ for 12-16 hours.

1.4 Extract the plasmid

Main experimental instrument:

Experiment equipment	Experiment reagent
Centrifugal machine	PTase bacterial liquid
Centrifuge tube	SP1、SP2、SP3
Adsorption column	Wash Solution
	Elution Buffer

Process:

1. Fill the centrifuge tube with PTase.
2. Put the centrifuge tube into the centrifuge at a speed of 8000xg for 2 min.
3. Take out the centrifuge tube and drop the supernatant from the top, only keeping the sediment below.
4. Use the micropipette to take 250 μL SP1 and put it into the centrifuge tube. And put the SP2 250ml and the 350ml SP3 into the tube and slowly shake it.
5. Then put it into the centrifuge at a speed of 8000xg for 2min.
6. Take the centrifuge out and use the micropipette into the adsorption column, which has a collection tube below.
7. Mix 96ul of absolute ethanol into the wash solution
8. Take out the centrifuge tube and transfer the clarified solution to the spin column (yellow tube) using a pipette set to 750 µL.
9. Place the spin column and collection tube into the centrifuge, set the speed to 8000 × g, and centrifuge for 30 seconds. Discard the liquid from the bottom of the collection tube.
10. Add 500 µL of Wash Solution to the spin column. Place the spin column and collection tube into the centrifuge, set the speed to 8000 × g, and centrifuge for 30 seconds. Discard the liquid from the bottom of the collection tube.
11. Repeat step 10.
12. Place the empty spin column into the centrifuge and centrifuge at 9000 × g for 1 minute to dry the membrane.
13. Prepare a new microcentrifuge tube and label it with the strain name. Insert the yellow spin column into the new tube. Add 75 µL of Elution Buffer directly onto the membrane, avoiding contact with the sides. Centrifuge at 9000 × g for 1 minute to elute the DNA.

1.5 PCR Procedure

Main experimental instrument

Experiment equipment	Experiment reagent
PCR Tubes	Primer solution
PCR machine	DDwater
Centrifugal machine	Primestar mix
Turbine mixer	DNA template

Process:

1. If precipitation is present in the primer solution, place the spin column in a centrifuge, set to 9000 × g, and centrifuge for 1 minute.
2. After centrifugation, add 100 µL dd water to the primer solution.
3. Take a PCR tube and label it with the primer name.
4. Add 25 µL Primestar Mix to the PCR tube.
5. Add 19 µL dd water to the PCR tube.
6. Add 2 µL forward primer (F-primer) to the PCR tube.
7. Add 2 µL reverse primer (R-primer) to the PCR tube.
8. Add 2 µL template DNA to the PCR tube.
9. Place the PCR tube on a vortex mixer and mix for 30 seconds.
10. Place the PCR tube in a centrifuge, set to 9000 × g, and centrifuge for 1 minute.
11. Load the prepared reaction system into the PCR machine for amplification.

PCR System Setup (Configuration)

Component	Volume (µL)	Component	Volume (µL)	Component	Volume (µL)	Component	Volume (µL)	Component	Volume (µL)	Component	Volume (µL)	Component	Volume (µL)
Mix	25	Mix	25	Mix	25	Mix	25	Mix	25	Mix	25	Mix	25
Gut1-F	2	Gut2-F	2	PTase-F	2	EPDH-F	2	pET-R	2	PK-F	2	TPL1-F	2
Gut1- R	2	Gut2- R	2	PTase-R	2	EPDH-R	2	pET-F	2	PK-R	2	TPL1-R	2
GUT1	2	GUT2	2	PTase	2	EPDH	2	PET	2	PK	2	TPL1	2
DDwater	19	DDwater	19	DDwater	19	DDwater	19	DDwater	19	DDwater	19	DDwater	19

PCR Program (Temperature Settings):

Temperature	Time
··98°C	10min
98°C	30ses
60°C	30ses
72°C	2min30sec
72°C	10min

Repeat steps 2–4 for 30 cycles.

1.6 Measure the concentrations of EPDH, Ptase, and PK using Nanadrop

Main experimental instrument

Experiment equipment	Experiment reagent
Nanodrop measurement cuvette	DDwater
	PK、 EPDH、PTase、PET

Process:

1. Turn on the Nanodrop spectrophotometer and calibrate the instrument.
2. Place the prepared sample into the Nanodrop measurement cuvette to measure the DNA concentration.
3. Use 2 µL of water to precisely contact the sample measurement area. Place the cuvette in the instrument and click to measure.
4. Here are the results:

EPDH: 57.1 ng/μL

PET: 81.8 ng/μL

PTase: 51.2 ng/μL

PTase: 57.3 ng/μL

PK: 20.1, 18.3 ng/μL

1.7 Gel Preparation

Main experimental instruments

Experiment equipment	Experiment reagent
Balance	Agarose
Erlenmeyer flask	TAE Buffer Solution
Microwave oven	Nucleic acid dye
Plastic mould manufacturing	Loading Buffer
Agarose gel electrophoresis tank and power supply	15K Marker
Turbine mixer	Buffer B2
Gel imaging system	Wash Solution
Knife	Elution Solution
Water bath	Homologous recombinase buffer
Centrifugal machine
Spin column
PCR Tubes

Process:

1. Turn on the balance, weigh 1g of agarose, and prepare two conical flasks.
2. Add 1 L of water and one packet of TAE powder into one conical flask, then mix the agarose with the TAE solution in the flask.
3. Take 100ml of the mixture and heat it in the microwave for 3 minutes at high temperature.
4. After heating, allow the mixture to cool to room temperature.
5. Add 4µL of nucleic acid dye to turn the solution pale pink for easier observation.
6. Pour the mixture into the prepared mold and let it cool for 15 minutes.
7. Prepare the agarose gel electrophoresis apparatus. Add 8 μL of loading buffer into the prepared PCR tubes.
8. Place the prepared materials in a vortex mixer, turn on the machine, and mix for 30 seconds.
9. Remove the prepared gel from the mold.
10. Place the gel into the electrophoresis tank with the sample wells facing the negative electrode.
11. Add TAE buffer until it covers the surface of the gel.
12. Load 20 μL of 15K marker into the sample wells, pipetting vertically.
13. Loading Order ⬇️
14. 20 μL marker
15. 50 μL GUT1
16. 50 μL GUT2
17. 50 μL TPL1
18. 50 μL PK
19. 50 μL EPDH
20. 50 μL PTase
21. 50 μL Bacteria P
22. Close the lid, set to 180V for 20 minutes.
23. Use a blade to cut out the successful bands.
24. Transfer the cut bands into a centrifuge tube and add 400 μL Buffer B2.
25. Place the gel under a gel imaging system to check if the results are successful.

Repeat the failed groups until imaging is successful.

1. Once all samples are successfully imaged, use a water bath to melt the gel.
2. Add the gel solution to a spin column, then centrifuge at 9000 × g for 30 seconds.
3. Discard the flow-through, add Wash Solution, centrifuge at 9000 × g for 30 seconds, and discard the flow-through again.
4. Repeat step 19 six times.
5. Dry the spin column by centrifuging at 9000 × g for 1 minute.
6. Transfer the spin column into a clean 1.5 mL microcentrifuge tube, then add 30 μL Elution Solution directly onto the center of the membrane.
7. Let the sample sit at room temperature for 1 minute, then centrifuge at 9000 × g for 1 minute. Store the eluted DNA solution.
8. Take 1 μL of the prepared solution and add it to the Nanodrop spectrophotometer for concentration measurement; for samples with a volume less than 1 μL, treat them uniformly as 1 μL during the operation.
9. Prepare two PCR tubes and label them with the names of the two plasmids.
10. Add 4 μL of homologous recombination enzyme buffer and 4 μL of the target gene fragment contained in the corresponding plasmid to each PCR tube. Vortex the mixture for 30 seconds, then incubate at 37°C for 30 minutes.

1.8 Transferring the PPE and GGT plasmid into Escherichia coli

Main experimental instruments:

Experiment equipment	Experiment reagent
Centrifuge tube	E. coli competent cells
Thermostat water bath	PPE、GGT
Centrifugal machine	LB medium
Temperature incubator	Spectinomycin hydrochloride
Culture dish	Ampicillin

Process:

1. Take two centrifuge tubes and add 100 μL of competent E. coli culture to each.
2. Add 10 μL of the target plasmid to each centrifuge tube and gently mix.
3. Incubate the centrifuge tubes in an ice bath for 30 minutes.
4. Perform a 45-second heat shock treatment on the centrifuge tubes to initiate the transformation process.
5. Immediately transfer the centrifuge tubes to an ice bath and cool for 3–5 minutes.
6. Add 900 μL of LB liquid medium to each centrifuge tube and mix thoroughly.
7. Incubate the centrifuge tubes at 37°C for 2 hours to complete bacterial recovery.
8. Pre-treatment of bacterial cultures: Centrifuge the bacterial cultures at 8000×g for 5 minutes to allow the bacterial cells to settle at the bottom of the centrifuge tube. 
9. Concentration and resuspension of bacterial suspension: Remove the supernatant, retain 100 μL of LB liquid medium to maintain bacterial cell viability, and gently resuspend the precipitate to form a uniform bacterial suspension.
10. Preparation of solid selective medium: 
11. Prepare LB solid medium containing the corresponding selection antibiotic (containing 1.5% agar powder): Mix LB liquid medium with the specific antibiotic, add 1.5% agar powder, heat to dissolve and mix thoroughly, then pour into 4 petri dishes and allow to cool and solidify naturally.
12. Antibiotic selection criteria: For the PK plasmid, spectinomycin hydrochloride is selected, matching its SmR resistance gene; for the GGT plasmid, ampicillin is selected, corresponding to its AmPt resistance gene. 
13. Spread the bacterial suspension evenly on the surface of the prepared selective medium, let it sit, then put it in an incubator. The next day, check the colony growth to see if the transformation was successful.

1.9 Extract PPE and GGT plasmids from single colonies on agar plates and perform PCR amplification

Main experimental instrument:

Experiment equipment	Experiment reagent
Biohazard safety equipment	Single colony
Culture dish	DDwater
PCR Tubes	Primer
Centrifugal machine
PCR machine

Process:

1. Transfer the petri dishes with single colonies into a biosafety cabinet and start sterile operation.
2. Use a micropipette to pick single colonies (10 colonies in total, covering 2 types of plasmids from 4 dishes), and transfer each into one of 14 PCR tubes.
3. Add 8 μl distilled water to each PCR tube to dissolve the colony.
4. Add 1 μl FR primers (upstream and downstream mixture), mix thoroughly by pipetting.
5. Place PCR tubes in a centrifuge and spin for 1 min (at standard speed, e.g., 5000×g, to collect liquid at the tube bottom).
6. Place PCR tubes into the PCR machine and run for 2.5 hours.

2.0 The target fragments PPE and GGT were verified by agarose gel electrophoresis

Main experimental instruments:

Experiment equipment	Experiment reagent
Balance	Agarose
Plastic mould manufacturing	TAE Buffer Solution
Electrophoresis tank and power supply	Nucleic acid dye
Ultraviolet imaging system	Loading Buffer
Metal bath	PCR amplification product
Culture tube	LB liquid medium
Biohazard safety equipment	SmR
Shaker	Amp⁺

Process:

1. Weigh 1 g of agarose and add to 100 ml of TAE buffer, heat until fully transparent (agarose dissolved).
2. Cool to 50–60℃, then add 3–5 μl nucleic acid dye and mix gently.
3. Pour the gel solution into an electrophoresis mold, let sit for 15 min until the gel is fully solidified.
4. Add 3 μl Loading Buffer into each PCR-amplified sample tube, mix thoroughly by pipetting.
5. Heat-treat for 3 min (e.g., boiling at 95℃ to denature nucleic acids, facilitating electrophoretic separation).
6. Load the treated samples into the wells of the agarose gel.
7. Start the electrophoresis apparatus, set the voltage to 180V, and run for 20 min.
8. After electrophoresis, transfer the gel to the UV imaging system and take photos to record band distribution.
9. Label culture tubes (distinguish between smr and ampt resistance groups), ensuring each group corresponds to the correct colony on the petri dish.
10. Add 3 mL LB liquid medium to each culture tube (operate under sterile conditions to avoid contamination).
11. According to a 1/1000 volume ratio, add 3 μl of SmR antibiotic to SmR group tubes and 3 μl of Amp⁺ antibiotic to Amp⁺ group tubes, gently invert 3 times to mix.
12. In the biosafety cabinet, use a micropipette to pick single colonies with the corresponding resistance.
13. Immerse the colony clump just below the surface of the liquid in the culture tube, gently pipette 3 times to disperse the colony and avoid floating.
14. Place culture tubes vertically in a 37℃ constant temperature shaker and begin shaking (if the device has a default speed, record as “220 rpm”; if no parameters are given, omit), incubate for 12–16 hours.
    

2.1 Culture Expansion and Protein Induction

To convert PK EPDH PTase and GUT1, GUT2, and TPl1 into E. coli BL21(DE3):

Main experimental instruments:

Experiment equipment	Experiment reagent
Bechtop	LB medium
Balance	SmR
Centrifuge tube	Amp+
Shaker	IPTG
Centrifugal machine	Lysis buffer
Turbine	Nickel column
Ultrasonic cell disrupto	Separation gel
Metal bath	Spacer gel
Spin Columns	Tris-Glycine-SDS buffer
Rubber sheets and combs	Loading Buffer
Electrophoresis tank and power supply	Marker
Temperature incubato

Process:

1. In the aseptic operation table, add 100 milliliters of LB medium to a conical flask, then add 100 microliters of SmR and PPE. Finally, seal the flask and write a label.
2. In the aseptic operation table, place 100 milliliters of LB medium into another conical flask, add 100 microliters of GGT and Amp+, seal it, and write a label.
3. In the aseptic operating table, place 20 milliliters of LB medium into another centrifuge tube, add 20 milliliters of PPE and SmR, seal it, and write a label.
4. In the aseptic operating table, place 20 milliliters of LB medium into another centrifuge tube, add 20 milliliters of PPE and SmR, seal it, and write a label.
5. Put the above-mentioned container into a 37-degree shaker and shake.
6. Take 4 large-capacity centrifuge tubes. Transfer the 4 bacterial cultures (cultured to OD₆₀₀ = 0.6-0.8, then add 100 μL 0.5mmol and 1mmol IPTG to induce protein expression, and incubate at 25°C with shaking for 12–16 hours) to the centrifuge tubes and fill them.

2.2 Protein purification and identification

Process:

1. Then centrifuge at 4°C and 12,000×g for 10 minutes.
2. After centrifugation, discard the supernatant, add 15 mL of lysis buffer to each centrifuge tube, gently resuspend the bacterial pellet using a 1 mL pipette, transfer the suspension to a vortex mixer, and then place it in an ice bath for 20 minutes.
3. Transfer the above-mentioned processed bacterial suspension to the ultrasonic cell disruptor for cell disruption.
4. Prepare 4 purification columns, labeled “Ppe 1mM,” “Ppe 0.5mM,” “Ggt 1mM,” and “Ggt 0.5mM,” respectively. Add 1 mL of nickel affinity resin to each purification column (to purify proteins by utilizing the specific binding of His tags to nickel ions).
5. Remove the sealing plug at the bottom of the purification column, wait for the liquid inside the column to flow out naturally, and retain the blue nickel affinity resin portion.
6. Take 4 new microcentrifuge tubes, label them according to the same rules as above, and set them aside for later use.
7. Transfer the ultrasonically disrupted bacterial suspension to a centrifuge tube and centrifuge at 10,000×g for 15 minutes. Collect the supernatant and transfer it to the corresponding microcentrifuge tube labeled in step 6.
8. Mix the supernatant (containing the target protein) from each microcentrifuge tube with the corresponding labeled purification column nickel affinity resin, and incubate at 4°C on a shaking incubator for 1.5-2 hours to remove impurities.
9. Prepare the separating gel: Mix 8.1 mL of ddH₂O, 6.7 μL of Acr-Bis (acrylamide-bisacrylamide), 5 mL of Gel Buffer A, 0.2 mL of APS (ammonium persulfate), and 12 μL of TEMED in sequence. After thorough mixing, inject the mixture into the gel casting apparatus and cover the upper layer with deionized water.
10. Prepare the concentrated gel: Mix 1.33 mL ddH₂O, 0.67 mL Acr-Bis, 2 mL Gel buffer B, 0.04 mL APS, and 0.004 mL TEMED in sequence, mix well, pour out the water on the upper layer of the separating gel in the gel preparation device, inject the concentrated gel, and insert the comb.
11. Once the gel has completely solidified, place it in the electrophoresis chamber and add Tris-Glycine-SDS buffer.
12. Prepare 20 microcentrifuge tubes, labeled as “Ppe 1mM 1-5,” “Ppe 0.5mM 1-5,” “Ggt 1 mM 1-5,” and “Ggt 0.5 mM 1-5.” Add the corresponding protein samples and loading buffer to each centrifuge tube, then heat in a 95°C metal bath for 10 minutes.
13. SDS-PAGE (Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis) analysis (for protein separation and identification)：
14. Add 15 μL of protein marker to the first sample well of the electrophoresis chamber, and add 20 μL of the above-treated protein samples (Ggt and Ppe series) to the remaining sample wells in sequence.
15. Cover and perform electrophoresis at a constant voltage of 120 V for 20 minutes.
16. After electrophoresis, remove the gel and place it on A4 paper for later use.

2.3 Convert SDS-PAGE to WB

Main experimental instruments:

Experiment equipment	Experiment reagent
Film transfer device	Transfer buffer solution
Sponge, filter paper	Lichun Red staining solution
Shaker	confining liquid
Chemiluminescence imaging system	TBST buffer solution
PVDF membrane	Chemiluminescence detection substrate
	primary antibodies
	second antibody

Process:

1. Place the cut 0.22 μm PVDF membrane in methanol and soak for 10-15 seconds (until it becomes translucent). Remove the blue protective film and rinse with deionized water, keeping the membrane wet throughout the process.
2. Prepare the transfer device: The transfer device has black and white sides. Place the gel in the “black adhesive, white membrane” direction to construct a sandwich structure (from the white edge to the black edge, in order: sponge → filter paper → PVDF membrane → gel → filter paper → sponge).
3. Remove the gel plate, take out the gel, and cut off the excess parts (retain the target band area, and avoid touching the gel surface with your hands during the operation); pre-soak the sponge in the pre-cooled transfer buffer solution, and place it on the transfer device blackboard (pay attention to the direction of the clamp).
4. Transfer the gel to the sponge, adjust the position to ensure that the area containing the bands completely covers the PVDF membrane (discard the part without bands), and avoid creating bubbles. Place the PVDF membrane (pay attention to the direction), flatten it, and remove any bubbles, then cover it with another layer of sponge and a lid, and clamp the film transfer device.
5. Place the sandwich structure into the transfer chamber (orientation: black side facing the black side of the chamber, white side facing the red side of the chamber), and fill with pre-chilled transfer buffer; place the transfer chamber in an ice box (maintain low temperature to prevent heat generated during transfer from affecting the results), and perform the transfer under one of the following conditions: Constant current 400 mA for 20 minutes, or constant voltage 100 V for 1 hour, or constant current 350 mA for 1.5 hours (adjust specific parameters based on buffer type and protein molecular weight).
6. After membrane transfer, remove the PVDF membrane (or nitrocellulose membrane, cellulose acetate membrane), immerse it in the Lichunhong staining solution, and agitate for 3 minutes (or until the bands are clear). Record the staining results and recover the staining solution. Rinse the membrane with distilled water or PBS 2–3 times, each for 1 minute, until the Lichun Red is mostly removed (if subsequent Western blot analysis is required, rinse until the bands disappear).
7. Immerse the PVDF membrane face up in blocking solution (5% skim milk powder, prepared with TBST) and incubate on a shaking incubator at room temperature for 1-2 hours; remove the blocking solution (can be recycled and reused).
8. Wash the membrane 2-3 times with TBST for 10 minutes each time; cut the target fragment from the membrane, transfer it to a container containing the primary antibody, incubate overnight at 4°C with slow shaking, and recover the primary antibody.
9. Wash the membrane three times with TBST for 10 minutes each time; add the secondary antibody, incubate on a shaking incubator at room temperature for 1.5 hours, and recover the secondary antibody.
10. Wash the membrane three times with TBST for 10 minutes each time; mix the two developers in a 1:1 ratio to prepare the chemiluminescence detection substrate working solution, immerse the membrane in it for 7-10 seconds, perform the entire process in the dark, and then perform exposure detection.

3.1 Conjugate the PPE plasmid and the GGT plasmid together into the competent cells of Escherichia coli

Main experimental instruments:

Experiment equipment	Experiment reagent
centrifuge tube	competent cell
thermostat water bath	LB medium
constant temperature shaker	SmR
centrifugal machine	Amp+
culture dish	PCR reaction buffer
temperature incubator	DDwater
PCR Tubes	primer
PCR machine	agarose
Agarose gel electrophoresis tank and power supply	TAE Buffer Solution
Plastic mould manufacturing	nucleic acid dye
Ultraviolet and visible spectrophotometer	Loading Buffer

Process:

1. Take the competent cells out of the ice.
2. Add 5 microliters of PPE and GGT plasmids, respectively, to the competent cells.
3. Place two small centrifuge tubes in a water bath at 42 degrees Celsius and heat for 45 seconds.
4. After heating, take it out quickly and let it stand in ice for four minutes
5. Add 900 microliters of LB medium to two small centrifuge tubes and gently shake.
6. Put it in a 37-degree shaker and shake for one and a half hours
7. After shaking, take it out and place it in a centrifuge for centrifugation at 8000xg for ten minutes
8. 8. Add 100 microliters of SmR and Amp⁺, respectively, to 100 milliliters of LB solution
9. 9. Pour an appropriate amount of the LB culture medium with antibiotics added into the culture dish
10. Repeat the above steps until all 500 milliliters of LB culture medium have been poured out.
11. After the centrifugation is completed in the centrifuge, discard the clear liquid on top, leaving 100 microliters. Blow up the colonies until the liquid becomes turbid.
12. 12. Take 30 microliters and evenly apply them to the petri dishes just used. Place them in a constant temperature incubator at 37 degrees Celsius and incubate upside down for 12 to 16 hours
13. Preparation of PCR reaction system: Take 8 PCR tubes, 4 of which are used for PPE detection and 4 for GGT detection. Add 10 μL of reaction buffer, 8 μL of ddH₂O, and 2 μL of the corresponding primers (PPE-specific primers or GGT-specific primers) to each PCR tube in sequence, then vortex mix.
14. Template bacterial suspension preparation: Select a single colony from the center of the culture plate. Divide one PPE-positive colony into two portions and add each to two PPE PCR tubes; divide one GGT-positive colony into two portions and add each to two GGT PCR tubes (with two replicates per group). Gently pipette to ensure the bacterial cells are evenly distributed throughout the reaction system.
15. PCR amplification verification: Place the above PCR tubes in a PCR machine to perform amplification reactions to verify the presence of the target gene.
16. Agarose gel preparation: Accurately weigh 1 g of agarose, add 100 mL of TAE buffer, heat and dissolve for 3 minutes, add 5 μL of nucleic acid dye and mix evenly, pour into the gel casting tray, and allow to solidify.
17. Gel electrophoresis analysis: After the PCR reaction is complete, add 3 μL of loading buffer to each reaction tube and centrifuge for 30 seconds to mix thoroughly. Place the solidified agarose gel into the electrophoresis chamber, add 20 μL of PCR product to each sample well, and perform electrophoresis at a constant voltage of 140 V for 20 minutes.
18. Culture and monitoring of bacterial suspension: Prepare 100 mL of LB culture medium, add 100 μL of a mixture of streptomycin sulfate (SMR) and ampicillin (AMP⁺) antibiotics, inoculate with 500 μL of co-transformed bacterial suspension, and take 1 mL of bacterial suspension as the initial sample. At regular intervals, take 1 mL of bacterial culture from different growth stages and measure its OD₆₀₀ value using a UV-visible spectrophotometer.
19. Carbon source regulation: Pre-prepare five groups of glucose-glycerol mixed carbon sources with different proportions. When thOD₆₀₀ value of the bacterial suspension reaches the preset threshold, add the corresponding proportions of glucose and glycerol to the culture system.

3.2 Growing curve

Main experimental instruments:

Experiment equipment	Experiment reagent
Ultraviolet and visible spectrophotometer	Culture medium of co-transformed strains

Process:

1. OD₆₀₀ values were measured at 0 h, 2 h, 4 h, 8 h, 12 h, 20 h, 24 h, 30 h, and 48 h of cultivation, with three replicates at each time point.
2. Based on the obtained data, a line chart was plotted (with cultivation time on the x-axis and erythritol concentration on the y-axis) to visually present the growth dynamics of the co-transformed strains.

3.3 HPLC identification of erythritol,Glycerol and Glucose

Main experimental instruments:

Experiment equipment	Experiment reagent
Centrifugal machine	5 mmol/L sulfuric acid solution
Filter plant
Binary High Pressure HPLC System
Glycerol
Glucose

Process:

1. We prepared five different fermentation media with varying substrate compositions and ratios (fermentation conditions: 37°C, pH 7-8), as follows: Glucose:Glycerol = 10:10 (g/L), Glucose:Glycerol = 20:10 (g/L), Glucose:Glycerol = 10:20 (g/L), Glucose:Glycerol = 20:0 (g/L), Glucose:Glycerol = 0:20 (g/L).
2. The bacteria were inoculated at 1% (v/v) in medium containing ampicillin (Amp) and streptomycin (SmR), and cultured until OD₆₀₀ reached 0.6-0.8, followed by induction with IPTG.
3. Samples were taken at different time points (5h, 16h, 24h, 48h) to measure the concentrations of glucose, glycerol, and erythritol.
4. After sampling, centrifuge the sample at 8000 r/min for 5 minutes to collect the supernatant.
5. Filter the collected supernatant through a 0.22 μm membrane.
6. Use an Aminex HPX-87H analytical column (300 mm × 7.8 mm, Bio-Rad, USA) coupled with a 2414 differential refractive index detector (Waters, USA) for analysis.
7. Set the detection conditions as follows: column temperature 35 °C, injection volume 10 μL, mobile phase 5 mmol/L sulfuric acid, and flow rate 0.6 mL/min.