Disposition of Culture

The Construction of Plasmid

2.1.Construction of pSCm-NFS1mu

A.Obtaining the gene fragment from pSCm-N20

Materials

• Bsal Enzyme
• pSCm-N20

Procedures

Use Enzyme Bsal to cut pSCm-N20 and get 5984 bp、441 bp, and 571 bp.

B.Agarose gel construction, deployment and recycle

Materials

• pipettor
• Casting Tray
• Microwave
• 1x TAE
• Agarose M
• 10000x Nucleic acid gel stain
• Erlenmeyer flask

Procedures

1)Dissolve 0.3g Agarose in 30mL Erlenmeyer flask

2)Add TAE (Running buffer)

3)Heat the material with a microwave

4)Melt the mixture at 30 sec intervals

5)See whether bubbles are forming if successfully mixed

6)Add 3μl nucleic acid gel stain (10000x)

7)Let it cool for 5~10 minutes

8)Pour the agarose mixture into the casting tray

9)Place the appropriate healthy comb to create the wells

10)Wait to solidify ~30 minutes

11)Remove the comb and place the gel in the gel box

C.Agarose gel electrophoresis

Materials

• Sample
• Marker
• Prepared gel (from the previous step)
• 10x Loading buffer (dye)
• Horizontal gel electrophoresis

Procedures

1)Extract 50µl of every sample; add 5µl of loading buffer (10x) to each.

2)Place the gel in the horizontal electrophoresis system

3)Extract 50µl of the mixed solution containing the sample and mix them till their colors are almost uniform

4)Add the marker to the first well and load 50µl of each sample into the wells

5)The procedure runs at 160V for 30min

6)Afterwards, take out the gel from the device and proceed to the following steps

D.DNA Gel Extraction

Materials

• agarose gel with 5984 bp
• clean scalpel
• 1.5mLEppendorf (EP) tube
• pipettes and sterilized pipette heads
• hot water bath
• buffer B2
• purification column and collection tube
• centrifuge
• wash solution containing pure ethanol

Procedures

1)Cut the slice of gel containing 5984 bp, cutting off as much unneeded gel as possible, and place it in a 1.5 mL Eppendorf (EP) tube.

2)Add 500 microliters of buffer B2 and put the tube into a 50°C hot water bath until the gel has completely melted.

3)Transfer the solution containing melted gel into a purification column that's in a collection tube and perform centrifugation at 8000 g for 30 seconds.

4)Empty the collection tube, put the purification column back in, and add 500 microliters of wash solution containing pure ethanol. Centrifugation at 9000 g was performed for 30 seconds, and the collection tube was emptied again.

5)Repeat above.

6)Perform centrifugation one more time at 9000 g for 1 minute, then open the cap of the tube and let it sit for one minute to allow the ethanol to evaporate.

7)Transfer the purification column to a 1.5 mL EP tube. Add 20 microliters of elution buffer at the center of the purification column.

8)Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 9000 g for 1 minute.

9)Discard the purification column. Store DNA in the EP tube at 4°C

E.Transferring the recombined fragment into DH5α

Materials

• Plasmid after ligation
E. coli in a competent state

Procedures

1) Ligated samples are added to E.Coli samples

2) Placed on ice for 20 minutes.

3) Heat at 42 ℃ for 45 seconds, then immediately put on ice for 2-3 minutes.

4) Add 900μL LB Agar solution and incubate for 30 minutes

5) Centrifuge at 5000xg for 3 minutes, extract 900μL of supernatant, then discard

6) Apply the end product to agar plates and incubate for 12-16 h at 37 degrees Celsius.

F.Colony PCR Validation

Materials

• PCR tube
• 2xmix
• C-SCm-gRNA-F
• N20-ISU1-R
• ddH2O
• LB cultural base
• Bacterial colonies
• PCR machine

Procedures

1)Prepare the system (20 μl: 10 μl ClonExpress Mix, 1 μl primer-F, 1 μl primer-R, 8ul ddH2O)

2)Pick a single colony with a 2.5 μl sterile pipette tip and perform aspiration and dispensing in the system.

3)Insert the mixture into a PCR thermal cycler and design the PCR program according to the manufacturer's instructions.

4)Perform agarose gel electrophoresis, and the presence of the correct bands indicates the successful construction of the plasmid pSCm-NFS1mu.

G.Extracting the plasmid

Materials

• Centrifuge tube
• Vortex
• Solution I
• Solution II
• Solution III
• Pipette
• Anhydrous ethanol
• Rinse solution
• Absorbent column
• Elution buffer
• Centrifuge

Procedures

1)Take 2 mL of bacterial culture and add it to a 2 mL centrifuge tube, centrifuge at 12000 rpm for 1 min and aspirate as much supernatant as possible.

2)Add 2 mL of bacterial culture to the same tube and centrifuge at 12000 rpm for 1 min; remove as much supernatant as possible.

3)Add 250 μL of Solution I to the centrifuge tube with the bacterial sediment and use a vortex shaker to suspend the bacterial cell sediment thoroughly.

4)Add 250μL of Solution II to the centrifuge tube and gently turn the tube up and down 6-8 times to fully lyse the bacteria.

5)Add 350μL of Solution III to the centrifuge tube and immediately mix well by gently turning the tube up and down 6-8 times. The centrifuge was at 12000 rpm for 10 min, and the supernatant was carefully transferred to another clean centrifuge tube using a pipette.

6)Take the supernatant from the previous step, add 0.35 times the volume of anhydrous ethanol and mix thoroughly.

7)Add the supernatant/mixing solution from the previous step into the adsorbent column (adsorbent column into the collection tube), let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorbent column back into the collection tube.

8)Add 750 μL of rinse solution to the column, centrifuge at 12000 rpm for 1 min, discard the waste solution, and put the column back into the collection tube.

9)Add 700 μL of rinse solution to the column, centrifuge at 12000 rpm for 1 min, discard the waste solution, and place the column in a collection tube.

10)Centrifuge at 12000 rpm for 2 min and leave the column open at room temperature for a few minutes.

11)Place the column into a clean centrifuge tube, add 40 drops of eluent to the centre of the adsorbent membrane, leave it at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min.

2.2.Construction of pSCm-ΔISU1

A. Construction of pSCm-ΔISU1

2.3.Construction of X-3-X

A. Construction of X-3-X

2.4.Construction of XI-2-XI

A. Construction of XI-2-XI

2.5.Construction of X-3-2XI

A. Construction of X-3-2XI

2.6.Construction of XI-2-2XI

A. Construction of XI-2-2XI

Yeast Transformation

3.1.xyl-8XI

We transferred the pHCas9-Nourse plasmid into the xyl-WT strain and screened the YPD-Nourse resistance plate to obtain xyl-Cas9 positive transformers. Then the X-3-XI-2-gRNA-HYG plasmid and the target gene fragment containing 2 copies of the PsXI expression frame were transferred into the xyl-Cas9 strain, and the positive invert was screened on the YPD-Nourse-HYG resistance plate. PsXI integration was then verified by colony PCR with primers GAP-XI-F1 / h-x-3d-bb-r1 and GAP-XI-F1 / h-xi-2d-bb-r1. We successfully integrated 8 copies of PsXI genes and named the strain xyl-8XI.

A. Preparation of S. cerevisiae competent cells.

Dip a sterile toothpick into a suitable amount of yeast liquid culture and streak it on a YPD solid agar plate to isolate single colonies. Pick a single colony and inoculate it into a 3 mL YPD liquid culture tube, then culture overnight at 30°C and 240 rpm on a shaker. Dilute the overnight culture 10 times and measure OD600, ensuring that OD600 is more significant than 0.2. Inoculate a 250 mL conical flask containing 25 mL of 2×YPD medium with an initial OD600 of 0.2, and culture at 30°C and 240 rpm until OD600 reaches 0.8 to 1.0. Centrifuge the culture at 20°C and 3000 rpm for 5 minutes, collect the cells, resuspend them in 25 mL of sterile water, and repeat the centrifugation step twice. Open a 42°C water bath and a 100°C metal bath. Resuspend the cells in 1 mL of sterile water, transfer the cell suspension to a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 30 seconds, discard the supernatant, collect the cells, and obtain competent yeast cells.

B. S. cerevisiae competent cell transformation method using LiAc.

Table 1. Reagents for LiAc conversion

Compositions Volume (μL)	Volume (μL)
PEG 3350（500 g/L）	240
LiAc（1 M）	36
Salmon sperm ssDNA（2 mg/L）	50
DNA + H2O	34

First, place 2 mg/L salmon sperm DNA in a 100°C metal bath for 5 minutes. After 5 minutes, quickly remove it and put it on ice for later use. Add the following components sequentially to the centrifuge tube containing the collected competent cells, following the order and quantities as shown in Table 1.

Perform a 42°C heat shock for 15 minutes, centrifuge at 12000 rpm for 30 seconds, discard the supernatant, add 1 mL YPD liquid medium, resuspend the cells, and seal with parafilm. Place it on a shaker, recover at 30°C and 240 rpm for 2 hours, then centrifuge at 3000 rpm for 4 minutes, discard 600 μL of the supernatant, resuspend, and spread 200 μL of the cell suspension evenly on a solid selection plate. Invert the plate and culture at 30°C for two days.

3.2.xyl-8XI-NFS1

After constructing strain xyl-8XI, we further mutated the nfs1 gene, mutated Ile at site 492 to Asn, and constructed strain XYL-8XI-NFS1. We introduced PGMC-GRNA-NFS1MU and the amplified homologous arm fragment into the prepared xyl-8XI receptive cells and cultured them at 30 °C. Subsequently, colony PCR was performed with primer NFS1 (1513-1535) -F/ NFS1-DN (2044-2072) -R. We successfully integrated 8 copies of the PsXI gene, and the Ile mutation at site 492 of the NFS1 gene was changed to Asn, and the resulting strain was named xyl-8XI-nfs1. The preparation of competent cells and the transformation method are consistent with the above.

3.3.xyl-8XI-ΔISU1

After the construction of strain xyl-8XI, we further performed gene knockout on the ISU1 gene to construct strain xyl-8XI-ΔISU1. We introduced pSCm-gRNA-ΔISU1 and the amplified homologous arm fragment into the prepared xyl-8XI receptive cells and cultured them at 30 °C. Colony PCR was performed with primer jd-ISU1 (282-308) -F/ jd-ISU1 (1209-1232) -R, and the final strain was named xyl-8XI-ΔISU1. We successfully integrated 8 copies of the PsXI gene and knocked out the ISU1 gene, and the resulting strain was named xyl-8XI-ΔISU1. The preparation of competent cells and the transformation method are consistent with the above.

3.4.xyl-8XI-nfs1-ΔISU1

After constructing strain xyl-8XI-nfs1, we further performed gene knockout on the ISU1 gene to construct strain XYL-8XI-NFS1-ΔISU1. We introduced pSCm-ΔISU1 and homologous arm fragments into xyl-8XI-nfs1 receptor cells and cultured them at 30 °C; Monoclonal colonies were selected for colony PCR verification using primer jd-ISU1 (282-308) -F/ jd-ISU1 (1209-1232) -R. We successfully integrated 8 copies of PsXI genes. The Ile at site 492 of the NFS1 gene was mutated into Asn, and the ISU1 gene was knocked out. The final strain was named wx-xyl-8XI-nfs1-ΔISU1. The preparation of competent cells and the transformation method are consistent with the above.

Functional Test

4.1.Solid medium assay for determining strains' xylose metabolism

Procedures

1)Primary: Pick one single colony and transfer it to a 3 mL YPDX test tube; add prokaryotic antibiotics, ampicillin and kanamycin to prevent infection of prokaryotic organisms in the test tube. Incubate for 24 h at 30℃, 240 rpm, and the OD600 should reach more than 10 (Note: Unless otherwise specified, each experiment consists of three replicates).

2)Secondary: Take 200 µL of primary seed bacterial solution and connect it to a fresh 3 mL YPD10X tube, incubate at 30℃, 240 rpm for 24 h. The culture was then incubated at 30℃ for 24 h.

3)Dispense fermentation medium YP40X: Dispense 5 mL/tube into sterile anaerobic tubes.

4)Calculate the inoculum: take the secondary seed broth and dilute it 20 times, measure the OD600 by spectrophotometer, and calculate the inoculum with the amount of 0.5g/L DCW; the calculation formula is: Inoculum volume(mL)=((Fermentation volume V(mL))/2)/(0.63×Seed cultureOD600)=0.5gDCW/L.

5)Inoculation: Take the calculated inoculum amount using the formula above of to the sterile 1.5 mL specification of the centrifuge tube.

6)1,2000 rpm centrifugation for 3 minutes.

7)Discard the supernatant wash with sterile water twice

8)1,2000 rpm centrifugation for 3 min, discard the supernatant.

9)Pipette 200 µL of culture medium from the above dispensed anaerobic tube into a 1.5 mL centrifuge tube, blowing it to make the yeasts float, and then transfer all of them back to the anaerobic tube and tighten the cap

10)Place the above inoculated anaerobic tube at 30℃, 240 rpm shaker for incubation and record the time.

4.2.Using HPLC to analyze the fermentation of strains

Procedures

1）The cellular biomass of the bacterium was detected by spectrophotometer, adjusting the wavelength to 600 nm, calibrating the zero with deionized water, and diluting the sample of bacterial liquid by 10-20 times to ensure that the OD600 of the spectrophotometric reading was in the range of 0 to 1.

2）Sample the fermentation broth every 8 hours by taking 200 µL of the culture into a 1.5 mL centrifuge tube. Use 100 µL for measuring OD600 and the remaining sample for HPLC analysis of xylose and ethanol until the end of fermentation.

3）The concentrations of xylose and ethanol were measured using an HPLC system (Waters e2695) equipped with an Aminex HPX-87H ion exchange column (300 × 7.8 mm; Bio-Rad) at 35°C and a Waters 2414 refractive index detector. Sulfuric acid (5 mM) served as the mobile phase with a flow rate of 0.6 ml min. The biological company conducted the specific testing.