With the continuous demand for petrochemical fuels driven by economic development, the reserves of such fuels on Earth are expected to be depleted in the future. Therefore, research on alternative energy sources is becoming increasingly important [1-3]. Currently, scholars have explored numerous potential alternative fuels, among which fuel ethanol stands out as one of the most promising substitutes for petrochemical fuels due to its unique characteristics, such as economic viability, renewability, and environmental friendliness. The production of fuel ethanol is derived from biomass, and the development of biomass energy globally has become a significant component of the new energy landscape. Using lignocellulosic biomass as raw material to produce second-generation fuel ethanol has become a mainstream direction for future development [4,5]. Therefore, it is essential to develop strains that can efficiently utilize both glucose and Xylose. This product aims to construct a yeast strain capable of synthesizing ethanol from Xylose at a much higher efficiency. To achieve this, we made four different strains by inserting 8 copies of the Xylose isomerase gene, introducing a mutation that enhances Xylose metabolism, and knocking out a gene that inhibits Xylose metabolism. Finally, we compared the Xylose metabolism efficiency of different strains to select the optimal genetic combination.

Fig. 1. The schematic diagram of Xylose metabolism

Design 1:

The gRNA plasmid pSCm-NFS1 is designed for the yeast strain where the NFS1 gene is mutated. The critical gene in the plasmid is the NFS1 gRNA, combined with the SNR52 promoter and SUP4 terminator. A mutation will later be induced on the NFS1 gene in the yeast to promote the metabolism of Xylose. It is constructed by connecting two N20 oligos and the gRNA backbone.

Fig. 2. The plasmid map of pSCm-NFS1

Build 1:

The pSCm-N20 plasmid was cut by BsaI to obtain 5984 bp, 441 bp and 571 bp bands, and 5984 bp was recycled on the gel as the backbone for backup.

Fig. 3. Gel electrophoresis of enzyme cutting of pSCm-N20 backbone

The two synthesised N20 oligo, gRNA-492I-F and gRNA-492I-R, annealed and self-propelled to form a double-stranded sequence using primer pairs, system: deionised water 35 µL, T4 ligase buffer 5 µL, primer gRNA-492I-F 5 µL (20 µM), and gRNA-492I-R 5 µL (20 µM). Hold at 95 °C for 5 min, decrease 5-10 °C per minute, hold at 16 °C for 10 min. Annealing products were diluted 10 times as fragments, and the gRNA backbone was ligated to the above fragments with a T4 ligase kit at 16 °C. The plasmid was then transformed into E. coli DH5α. LB plates containing a final concentration of 100 µg/mL ampicillin antibiotic were coated and incubated overnight at 30 °C until there were transformants. Figure 4 shows the presence of single colonies on the plate.

Fig. 4. Transformation plate of pSCm-NFS1

A number of transformants were randomly picked and was verified by colony PCR using the primers c-SCm-gRNA-F/gRNA-492I-R. The correct length of 288 bp was obtained. Figure 5 shows the band consistent with the target size.

Fig. 5. Gel electrophoresis validation of pSCm-NFS1

The transformants with the correct length were transferred to LB test tubes containing 100 µg/mL of ampicillin antibiotic, cultured at 30 °C, 220 rpm overnight, and the plasmid was extracted.

The plasmid was sent for sequencing, and the primer for sequencing was c-SCm-gRNA-F, and 1 reaction was measured. According to the results shown in Figure 6, the parts are successfully ligated, confirming the successful construction of the pSCm- NFS1 plasmid.

Fig. 6. Sequencing map of pSCm-NFS1

Learn 1:

The pSCm-NFS1 plasmid is constructed for the mutation of the NFS1 gene in the yeast using homologous recombination to promote Xylose metabolism capability.

Design 2:

The key gene in the pSCm-△ISU1 plasmid is ISU1 gRNA, with SNR52 promoter and SUP4 terminator. It is constructed by connecting two N20 oligos and the gRNA backbone.

Fig.7. The plasmid map of pSCm-△ISU1

Build 2:

The two synthesised N20 oligos, N20-ISU1-F and N20-ISU1-R, were first annealed and self-ligated to form a double-stranded sequence. system: deionised water 35 µL, T4 ligase buffer 5 µL, primers N20-ISU1-F 5 µL (20 µM), N20-ISU1-R 5 µL (20 µM). Hold at 95 °C for 5 min, decrease the temperature by 5-10 °C per minute and hold at 16 °C for 10 min. The annealed product was diluted 10-fold as a fragment, and the gRNA backbone was chemically transformed to DH5α by ligating the above fragment with the T4 ligase kit at 16 °C in vitro. LB plates containing a final concentration of 100 µg/mL ampicillin antibiotic were coated and cultured in an incubator at 30 °C overnight until there were transformants. Figure 8 shows the presence of single colonies on the plate.

Fig. 8. Transformation plate of pSCm-△ISU1

A number of transformants were randomly picked and were verified by colony PCR using the primers c-SCm-gRNA-F / N20-ISU1-R. The correct length of 288 bp was obtained. Figure 9 shows the band consistent with the target size.

Fig. 9. Gel electrophoresis validation of pSCm-△ISU1

The transformants with the correct length were transferred to LB test tubes containing 100 µg/mL of ampicillin antibiotic, cultured at 30 °C, 220 rpm overnight, and the plasmid was extracted and sequenced. The sequencing primer was c-SCm-gRNA-F, and 1 reaction was measured. According to the results shown in Figure 10, the parts are successfully ligated, confirming the successful construction of the pSCm-△ISU1 plasmid.

Fig. 10. Sequencing map of pSCm-△ISU1

Learn 2:

The pSCm-△ISU1 plasmid is constructed for the knockout of the ISU1 gene in the yeast using homologous recombination. In order to promote the Xylose metabolism capability of yeast.

Design 3:

X-3-XI is constructed by integrating the PsXI gene into the X-3 site. A biotech company provides the backbone plasmid of the X-3 integration site. The PsXI gene is joint by TEF1 promoter and ADH1 terminator.

Fig. 11. The plasmid map of X-3-XI

Build 3:

The pHCas9 plasmid was used as a template, and primer pair TEF1p-F1/ TEF1p-XI-R1 amplified the TEF1 promoter with a size of 430bp; the FDP-PsXI plasmid containing the PsXI gene was used as a template, and primer pair XI-TEF1p-F1/ XI-ADH1t-R1 amplified the PsXI gene sequence with a size of 1,354bp. The brewer's yeast colonies or genome as a template, primer pair ADH1t-XI-F1/ ADH1t-R1 amplified ADH1 terminator with a size of 214bp. Figure 12 shows the band consistent with the target size, indicating successful amplification.

Fig. 12. Gel electrophoresis validation of gene amplification of TEF1 promoter, PsXI, and ADH1 terminator

The above three fragments were processed by overlap PCR using primer TEF1p-F1/ ADH1t-R1. The size of the fragment was 1920bp. Figure 13 shows the band consistent with the target size, indicating the overlap PCR is successful.

Fig. 13. Overlap PCR results of TEF1-PsXI-ADH1

The 1920 bp fragment was recycled and ligated into the PsXI gene sequence, also using the primer TEF1p-F1/ Xho1. The 1920bp fragment was recycled using Sgs1+Xho1 double digestion and ligated into the X-3 locus integration backbone plasmid. It was also double digested using Sgs1+Xho1. Figure 14 shows the band consistent with the target size. The enzyme cutting is successful.

Fig. 14. Results of enzyme cutting of X-3 backbone and TEF1-PsXI-ADH1

The plasmid is transformed into DH5α and cultivated on LB-Amp plates. Figure 15 shows the presence of single colonies on the plate.

Fig .15. Transformation plate of X-3-XI

Colonial PCR was carried out using the primers grna-HYG-baseplasmid-seq-7/ XI-ADH1t-R1. The target band was 2065bp. Figure 16 shows the band consistent with the target size.

Fig. 16. Gel electrophoresis validation of X-3-XI

Sequence using the primers grna-HYG-baseplasmid-seq-7 and h-x-3d-bb-r1. According to the results shown in Figure 17, the construction of the X-3-XI plasmid is successful.

Fig. 17. Sequencing map of X-3-XI

Learn 3:

X-3-XI plasmid is prepared for a second round of PsXI gene integration into the X-3-XI to produce a plasmid with two copies of the PsXI gene, which would result in 8 copies of the gene in the yeast.

Design 4:

PCR was used to obtain the backbone XI-2 and the target gene TEF1-PsXI-ADH1, followed by a link between the XI-2 backbone and the target gene TEF1-PsXI-ADH1.

Figure 18. The plasmid map of XI-2-XI

Build 4:

We constructed a plasmid XI-2-XI containing a single copy of the XI gene. We amplified and validated the backbone XI-2 and the target gene TEF1-PsXI-ADH1. The results in Figure 19 showed matching band sizes, indicating successful amplification. We ligated the XI-2 backbone and the target gene TEF1-PsXI-ADH1.

Figure 19. Validation result of linear pX-3, linear pXI-2, and TEF1-PsXI-ADH1

We ligated the XI-2 backbone and the target gene TEF1-PsXI-ADH1 and transformed it into competent E.coli DH5α. Figure 20 shows the results after culturing E. coli, where single colonies can be observed.

Figure 20. XI-2-XI colonies

We performed colony PCR to validate the cultured colonies. Figure 21 displays the results of the colony PCR, showing bands of approximately 2065 bp, consistent with the expected fragment size, validating our successful transformation and plasmid construction.

Figure 21. Validation of colony PCR of XI-2-PsXI

The colonies were also sent for sequencing. According to the results shown in Figure 22, the TEF1-PsXI-ADH1 gene was successfully ligated to the backbone without any apparent mutations, confirming the successful construction of the XI-2-XI plasmid.

Figure 22. Sequencing result of XI-2-XI

Learn 4:

Based on the construction of the X-3-XI plasmid, the newly constructed XI-2-XI plasmid has its integration site modified to XI-2.

Design 5:

A biotech company synthesized the target gene, XI. Obtain the promoter GAP, the coding gene PsXI, and the terminator PsXI separately. Perform overlap PCR to connect these three fragments and finally ligate them to the linearized vector.

Figure 23. The plasmid map of X-3-2XI

Build 5:

We constructed a plasmid X-3-2XI containing two copies of the XI genes. Figure 24 shows the PCR validation results for the promoter GAP, the coding gene PsXI, and the terminator CYC1, with band sizes matching the expected lengths of 693 bp, 1350 bp, and 275 bp, respectively, indicating successful amplification.

Figure 24. Amplification result of GAP pro, PsXI, and CYC1 ter

Overlap PCR was performed on these fragments. Figure 25 shows the results of the overlap PCR, with a band size consistent with the expected length of 2245 bp, indicating successful synthesis of the target gene.

Figure 25. Overlap PCR result of GAP-PsXI-CYC1

We amplified and validated the backbone X-3-XI and the target gene GAP-PsXI-CYC1. The results in Figure 26 showed matching band sizes, indicating successful amplification.

Figure 26. Validation of X-3-XI, XI-2-XI, and GAP-PsXI-CYC1

We ligated the X-3-XI backbone and the target gene GAP-PsXI-CYC1 and transformed it into competent E.coli DH5α. Figure 27 shows the results after culturing E. coli, where single colonies can be observed.

Figure 27. X-3-2XI colonies

We performed colony PCR to validate the cultured colonies. Figure 28 displays the results of the colony PCR, showing bands of approximately 904 bp, consistent with the expected fragment size, validating our successful transformation and plasmid construction.

Figure 28. Validation of colony PCR of X-3-2XI

The colonies were also sent for sequencing. According to the results shown in Figure 29, the GAP-PsXI-CYC1 gene was successfully ligated to the backbone without any apparent mutations, confirming the successful construction of the X-3-2XI plasmid.

Figure 29. Sequencing result of X-3-2XI

Learn 5:

Based on the construction of the X-3-XI plasmid, the newly constructed X-3-2XI plasmid obtained two copies of the XI gene.

Design 6:

PCR was used to obtain the backbone XI-2-XI and the target gene GAP-PsXI-CYC1, followed by a link of the XI-2-XI backbone and the target gene GAP-PsXI-CYC1.

Figure 30. The plasmid map of XI-2-2XI

Build 6:

We constructed a plasmid XI-2-2XI containing two copies of the XI genes. We amplified and validated the backbone XI-2-XI and the target gene GAP-PsXI-CYC1. The results in Figure 31 showed matching band sizes, indicating successful amplification.

Figure 31. Validation result of X-3-XI, XI-2-XI, and GAP-PsXI-CYC1

We ligated the XI-2-XI backbone and the target gene GAP-PsXI-CYC1 and transformed it into competent E.coli DH5α. Figure 32 shows the results after culturing E. coli, where single colonies can be observed.

Figure 32. XI-2-2XI colonies

We performed colony PCR to validate the cultured colonies. Figure 33 displays the results of the colony PCR, showing bands of approximately 904 bp, consistent with the expected fragment size, validating our successful transformation and plasmid construction.

Figure 33. Validation of colony PCR of XI-2-2XI

The colonies were also sent for sequencing. According to the results shown in Figure 34, the GAP-PsXI-CYC1 gene was successfully ligated to the backbone without any apparent mutations, confirming the successful construction of the XI-2-2XI plasmid.

Figure 34. sequencing result of XI-2-2XI

Learn 6:

Based on the construction of the X-2-XI plasmid, the newly constructed XI-2-2XI plasmid obtained two copies of the XI gene.

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