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

BBa_K5351007 (X-3-2XI)

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.

Figure 2: Amplification result of GAP pro, PsXI, and CYC1 ter

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.

Figure 3: Overlap PCR result of GAP-PsXI-CYC1

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.

Figure 4: Validation of X-3-XI, XI-2-XI, and GAP-PsXI-CYC1

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.

Figure 6: Validation of colony PCR of X-3-2XI

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

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, as shown in Table 1, indicate a significant improvement in ethanol production capacity in the engineered strains. Notably, our strain xyl-8XI-nfs1 exhibited a 5.2-fold increase in ethanol production compared to the parental strain, reaching 662.18 mg/L. This result further validates the success of our genetic modifications to the strains.

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.