Part:BBa_K5351001

pSCm-NFS1mu

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1135
  Illegal NheI site found at 4384
  Illegal NotI site found at 2401
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 7707
  Illegal BglII site found at 7904
  Illegal BamHI site found at 2246
  Illegal BamHI site found at 2357
  Illegal XhoI site found at 2408
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Origin

Synthesized by the team and constructed as a gRNA plasmid for yeast.

Properties

This part is designed to mutate the NFS1 gene in yeast, enhancing the metabolism of xylose, an essential step for efficient ethanol production.

Usage and Biology

pSCm-NFS1mu is a gRNA plasmid designed to induce a mutation in the NFS1 gene in yeast. The mutation promotes xylose metabolism, making this part significant for biofuel production. The design of the plasmid includes the SNR52 promoter and SUP4 terminator, which regulate the expression of gRNA targeting the NFS1 gene.

Experimental Approach

Figure 1. Plasmid map of pSCm-NFS1.

The pSCm-N20 plasmid was digested using BsaI, generating fragments of 5984 bp, 441 bp, and 571 bp. The 5984 bp fragment was used as the backbone for constructing the gRNA plasmid.

Figure 2. Gel electrophoresis of pSCm-N20.

The N20 oligos gRNA-492I-F and gRNA-492I-R were synthesized and annealed to form a double-stranded sequence. The gRNA backbone was ligated to the fragments, and the resulting plasmid was transformed into *E. coli* DH5α.

Figure 3. Transformation plate of pSCm-NFS1.

Colonies were verified through colony PCR, and the correct fragment length of 288 bp was obtained.

Figure 4. Gel electrophoresis validation of pSCm-NFS1.

Sequencing confirmed the successful construction of pSCm-NFS1, showing the correct sequence.

Figure 5. Sequencing map of pSCm-NFS1.

The pSCm-NFS1 plasmid was introduced into yeast strains to induce mutations, leading to enhanced xylose metabolism.

Figure 6. PCR verification of mutated strains.

Xylose metabolism capabilities were evaluated through a solid medium assay. Mutated strains demonstrated significantly improved xylose utilization compared to wild-type strains.

Figure 7. Xylose metabolism plate assay.

Fermentation experiments were conducted to measure ethanol production. High-performance liquid chromatography (HPLC) showed that the NFS1 mutant strain produced significantly more ethanol compared to the wild-type strain.

Figure 8. HPLC results of ethanol production.

Figure 9. Colony PCR verification of xylose metabolism strains.

The xylose utilization and ethanol production of the engineered strains were further validated through fermentation assays.

Figure 10. Growth and ethanol production of xylose metabolism strains.

The results confirm the success of genetic modifications to enhance xylose metabolism in yeast.

Figure 11. Comparison of xylose metabolism and ethanol production.

Reference

Wei F., Li M., Wang M., et al. (2020). A C6/C5 co‐fermenting *Saccharomyces cerevisiae* strain with the alleviation of antagonism between xylose utilization and robustness. *GCB Bioenergy*, 13(1), 83–97.

Brat D., Boles E., Wiedemann B. (2009). Functional expression of a bacterial xylose isomerase in *Saccharomyces cerevisiae*. *Applied Microbiology and Biotechnology*, 75, 2304-2311.