Part:BBa_K5351007

X-3-2XI

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 3656
  Illegal NotI site found at 4104
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 3877
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 5120
  Illegal AgeI site found at 6434
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2651
  Illegal BsaI.rc site found at 132
  Illegal SapI site found at 284
  Illegal SapI.rc site found at 1629
  

Construction Design

We constructed a plasmid X-3-2XI (BBa_K5351007) containing two copies of the XI genes. The integration site is X-3 (BBa_K4845003). 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.

Fig. 1. The plasmid map of X-3-2XI

Engineering Principle

We constructed a plasmid X-3-2XI (BBa_K5351007) containing two copies of the XI genes. The integration site is X-3 (BBa_K4845003).

Experimental Approach

We constructed a plasmid X-3-2XI containing two copies of the XI genes. Figure 2 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.

Fig. 2. Amplification result of GAP pro, PsXI, and CYC1 ter

Overlap PCR was performed on these fragments. Figure 3 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.

Fig. 3. 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 4 showed matching band sizes, indicating successful amplification.

Fig. 4. 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 5 shows the results after culturing E. coli, where single colonies can be observed.

Fig. 5. X-3-2XI colonies

We performed colony PCR to validate the cultured colonies. Figure 6 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.

Fig. 6. Validation of colony PCR of X-3-2XI

The colonies were also sent for sequencing. According to the results shown in Figure 7, 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.

Fig. 7. Sequencing result of X-3-2XI

Characterization/Measurement

1. Yeast Transformation

a) xyl-8XI

First, 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. Agarose gel electrophoresis results (Figure. 8) showed that the target band expanded 900 bp bands, as expected, indicating that we successfully integrated 8 copies of PsXI genes and named the strain xyl-8XI.

Fig. 8. PCR and colony map of xyl-8XI

b) 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. The result of agarose gel electrophoresis (Figure. 9) showed that the target band was amplified by about 495 bp, as expected, indicating that 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.

Fig. 9. PCR validation diagram and colony diagram of xyl-8XI-nfs1

c) 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. The results of agarose gel electrophoresis (Figure 10) showed that the target band was amplified by about 451 bp, as expected, indicating that we successfully integrated 8 copies of the PsXI gene and knocked out the ISU1 gene.

Fig. 10. PCR and colony map of xyl-8XI-ΔISU1

d) 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. Results of agarose gel electrophoresis (Figure 11) showed that the target band was amplified by about 451 bp, as expected, indicating that 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.

Fig. 11. Colony PCR and colony map of xyl-8XI-nfs1-ΔISU1

2. Functional Test

a) Solid Medium Assay for Determining Strains' Xylose Metabolism

The strains xyl-8XI, xyl-8XI-NFS1, xyl-8XI-ΔISU1, xyl-8XI-NFS1-ΔISU1, and XYL-8XI-NFS1-ΔISU1 were evaluated for their xylose utilization capabilities, and their growth performances were compared. The xylose plate test results (Figure 12) revealed that the XYL-8XI-NFS1 strain exhibited robust growth even under xylose dilutions of 10-12, surpassing the performance of xyl-8XI, xyl-8XI-ΔISU1, and xyl-WT strains. This observation suggests that the mutation of the NFS1 gene positively influenced xylose metabolism.

Fig. 12. The xylose plate detected xylose metabolism.

b) Using HPLC to Analyze the Fermentation of Strains

High-performance liquid chromatography (HPLC) testing will further quantitatively analyze the xylose metabolism to verify the actual performance of different strains in xylose metabolism. The strains were fermented in a YPDX medium to evaluate their actual xylose metabolism efficiency. The results of HPLC will further support the advantages of NFS1 mutant strains in xylose metabolism, providing new ideas and a theoretical basis for yeast strains to optimize xylose utilization.

The concentrations of xylose 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⁻¹ [1]. The biological company conducted the specific testing.

From Figure 13, it is evident that introducing xylose isomerase into different parent strains resulted in enhanced xylose metabolism capabilities. Further validation of the NFS1 mutant strains' advantages in xylose metabolism was achieved through the actual xylose metabolism performance in the fermentation broth. The strain with the fastest xylose utilization rate nearly depleted the xylose in the medium within 40 hours. Additionally, observations on strain growth revealed that the modified strains exhibited minimal impact on growth, with an earlier onset of exponential growth. This rapid growth of the strains also positively influenced xylose utilization.

Fig. 13. Comparison of xylose metabolism and growth status of the modified strains (a: Xylose metabolism profiles of different strains; b: Growth status comparison among different strains)

Conclusion

The ultimate goal of enhancing xylose utilization in our engineered strains is for ethanol production. Therefore, we also measured the ethanol content in the fermentation broth after fermentation to assess the potential of our strains in the field of second-generation biofuel ethanol production. Experimental results indicate a significant improvement in ethanol production capacity in the engineered strains, validating the success of our genetic modifications.

Strains	Ethanol concentration (mg/L)
xyl	106.72
xyl-8XI	298.74
xyl-8XI-nfs1	662.18
xyl-8XI-△ISU1	367.16
xyl-8XI-nfs1-△ISU1	307.10

Discussion

In our experiment, we utilized YPDX medium rather than glucose and xylose derived from lignocellulosic biomass hydrolysate. Therefore, in the future, we will need to ferment with actual hydrolysate, which contains not only glucose and xylose but also a significant amount of inhibitors generated from lignin decomposition. This poses specific requirements for the stress tolerance of our strains [2,3]. We aim not only to enhance the strains' xylose utilization capability but also to increase their robustness in the hydrolysate, ultimately facilitating improved production of second-generation biofuel ethanol.

References

[1] Wei Fangqing, Li Menglei, Wang Ming, et al. A C6/C5 co-fermenting Saccharomyces cerevisiae strain with the alleviation of antagonism between xylose utilization and robustness. GCB Bioenergy, 2020, 13(1): 83–97.
[2] Brat D, Boles E, Wiedemann B. Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 2009, 75: 2304-2311.
[3] Liu CG, Xiao Y, Xia XX, et al. Cellulosic ethanol production: Progress, challenges and strategies for solutions. Biotechnol Adv, 2019, 37(3): 491-504.