Results
1. Aspergillus niger derived xylanase expression plasmid construction
Figure 1 pXlnB and pXylan-B plasmids map
To utilize the xylan component contained in the wheat B starch, we cloned the xylanase expression gene from Aspergillus niger. The xylanase expression cassette contained pXlnB plasmid was constructed firstly to prepare the final plasmid pXylan-B (Figure 1).
Figure 2 Plasmids construction used fragments PCR amplification. (A) Lane 1: GAP promoter, 695 bp. Lane 2: AnXlnB CDS, 706 bp. Lane 3: CYC1 terminator, 276bp. Lane 4: pXlnB plasmid backbone fragment, 1757 bp. Lane 5: TPI1 promoter, 614 bp. Lane 6: AnXlnD CDS, 2443 bp. Lane 7: pXlnD plasmid backbone fragment, 1804 bp. (B) Lane 1: pXlnB plasmid backbone fragment, 1804 bp. Lane 2: pXlnD plasmid backbone fragment, 1804 bp. (C) Lane 1: pXylan-B plasmid backbone, 5479 bp. Lane 2: pXylan-BD plasmid backbone, 5479 bp.
For the pXlnB plasmid construction, the promoter GAP, codon-optimized AnXlnB CDS, and CYC1 terminator PCR bands were shown in the Figure 2A, lane 1, lane2, and lane 3, respectively. The AnXlnB expression cassette was obtained through the overlap PCR. The backbone fragment (kanR with ori) was amplified using two round PCR, the first round and the final fragment band were shown in Figure 2A lane 4 and Figure 2B lane 1, respectively. The backbone was cut with Bsa1 restriction enzyme and ligated with the AnXlnB expression cassette to make the plasmid pXlnB.
For the construction of the final plasmid pXylan-B, the pXlnB was cut with Sap1 restriction enzyme, and the backbone part (Figure 2C) was also cut with the same enzyme, these two parts were ligated to make the final plasmid pXylan-B.
2. Aspergillus niger derived xylanase and β-xylosidase expression plasmid construction
Figure 3 pXlnD and pXylan-BD plasmids map
For the effective utilization of the xylan component present in the wheat B starch, the Aspergillus niger derived β-xylosidase (Figure 3A) was also cloned together with the xylanase expression gene. To make the final plasmid pXylan-BD (Figure 3B), the pXlnD plasmid was constructed firstly. The promoter TPI1, codon-optimized AnXlnD CDS, and CYC1 terminator PCR bands were shown in the Figure 2A, lane 5, lane6, and lane 3, respectively. The AnXlnD expression cassette was obtained through the overlap PCR. The backbone fragment (kanR with ori) was amplified using two round PCR, the first round and the final fragment band were shown in Figure 2A lane 7 and Figure 2B lane 2, respectively. The backbone was cut with Bsa1 restriction enzyme and ligated with the AnXlnD expression cassette to make the plasmid pXlnD.
For the construction of the final plasmid pXylan-BD, the pXlnB and pXlnD were both cut with Sap1 restriction enzyme, and the backbone part (Figure 2C) was also cut with the same enzyme, these three parts were ligated to make the final plasmid pXylan-BD.
Figure 4 Positive colonies verification through the colony PCR. Lane 11 to 20, pXlnB plasmid colony PCR verification. Lane 21 to 30, pXlnD plasmid colony PCR verification.
Figure 4 demonstrated the positive colonies verification of the plasmids pXlnB and pXlnD. The number of 12 to 16, 18 to 20 were the positive colonies of the plasmid pXlnB, the number of 21, 23, 24, 27 to 30 were the positive colonies of the plasmid pXlnD. Number 12 of pXlnB and number 23 of pXlnD were sent for the sequencing.
Figure 5 Plasmids DNA sequencing. A: pXlnB plasmid. B: pXlnD plasmid.
The positive transformants verified using the colony PCR were sent for the DNA sequencing, as shown in Figure 5, both the plasmids pXlnB and pXlnD were constructed successfully.
Figure 6 Positive colonies verification through the colony PCR (A) and DNA sequencing of the plasmid pXylan-B (B) and pXylan-BD (C).
Figure 6A demonstrated the positive colonies verification of the plasmids pXylan-B and pXylan-BD. The numbers 10, 12, and 14 were the positive colonies of the plasmid pXylan-B, the numberss 2, 4, and 6 were the positive colonies of the plasmid pXylan-BD. Number 12 of pXylan-B and number 4 of pXylan-BD were sent for the sequencing. Figures 6B and C showed that both the pXylan-B and pXylan-BD plasmids were constructed successfully.
3. The xylan utilization Saccharomyces cerevisiae strain construction and fermentation test
Figure 7 Fermentation performance of the plasmids transformed S. cerevisiae strains in the simulated wheat B starch medium. A: OD value. B: Sugar concentration.
The plasmids pXylan-B and pXylan-BD were transformed into the S. cerevisiae strain, respectively. The resulting positive transformants were used in the fermentation test. In the simulated wheat B starch medium (YPD20Xylan20), all the strains showed almost the same growth performance during the first 8 h, this is due to the strains preferentially utilized the glucose present in the media. This was verified again in Figure 7B, all the strains showed the comparable sugar utilization capacity, the xylan utilization ability may be covered by the glucose. Therefore, to verify the strains’ xylan utilization capacity, a xylan as the sole carbon source medium was essential in further study.
The sugar consumption data showed that starting from 2 hours, the WXA/pXylan-B and WXA/pXylan-BD strain was slightly higher than the WXA control, which could be interpreted as decomposing xylan and producing reducing sugars. Therefore, the engineered bacteria we constructed can decompose the xylan successfully.
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