Part:BBa_K5351001
pSCm-NFS1mu
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1135
Illegal NheI site found at 4384
Illegal NotI site found at 2401 - 21INCOMPATIBLE 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 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Construction Design
The gRNA plasmid pSCm-NFS1mu (BBa_K5351001) is designed for the yeast strain where the NFS1 (BBa_K5351002) 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.
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.
Engineering Principle
The gRNA plasmid pSCm-NFS1mu (BBa_K5351001) is designed for the yeast strain where the NFS1 (BBa_K5351002) 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.
Experimental Approach
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.
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.
A number of transformants were randomly picked and verified by colony PCR using the primers c-SCm-gRNA-F/gRNA-492I-R. The correct length of 288 bp was obtained. Figure 4 shows the band consistent with the target size.
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 5, the parts are successfully ligated, confirming the successful construction of the pSCm-NFS1 plasmid.
Characterization/Measurement
The pSCm-NFS1 plasmid is constructed for the mutation of the NFS1 gene in the yeast using homologous recombination to promote Xylose metabolism capability.
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 (Fig. 6) 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.
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 agar-gel electrophoresis (Fig. 7) 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.
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 agar-gel electrophoresis (Fig. 8) showed that the target band was amplified by about 451bp, as expected, indicating that we successfully integrated 8 copies of the PsXI gene and knocked out ISU1 gene, and the resulting strain was named 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 agar-gel electrophoresis (Fig. 9) showed that the target band was amplified by about 451bp, 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.
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 (Fig. 10) 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. Specifically, xylose metabolic capacity was ranked as follows: XYL-8XI-NFS1 > XYL-8XI-NFs1-ΔISU1 > xyl-8XI > XYL-8XI-ΔISU1 > XYL-8XI-ΔISU1 > xyl-WT. These results indicated that NFS1 gene mutation significantly improved xylose metabolic capacity, while ISU1 gene knockout had a specific effect on xylose metabolism, but it was not as significant as NFS1 mutation. Through this experiment, we have identified promising targets for xylose metabolism enhancement.
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, and these results provide 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 Fig 11, 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.
Ethanol Production
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.
Table 1. The ethanol production levels of different strains (48 h)
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
- 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 [J]. GCB Bioenergy, 2020, 13(1): 83–97.
- Brat D, Boles E, Wiedemann B. Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae [J]. Applied Microbiology and Biotechnology, 2009, 75: 2304-2311.
- Liu CG, Xiao Y, Xia XX, et al. Cellulosic ethanol production: Progress, challenges and strategies for solutions [J]. Biotechnol Adv, 2019, 37(3): 491-504.
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