Part:BBa_K3996005

AnXlnD orf

AnXlnD orf

Usage and Biology

This protein is involved in the pathway xylan degradation, which is part of Glycan degradation. Xylan 1,4-beta-xylosidase involved in the hydrolysis of xylan, a major structural heterogeneous polysaccharide found in plant biomass representing the second most abundant polysaccharide in the biosphere, after cellulose.

Construct design

Figure 1. pXlnD and pXylan-BD plasmids map..

we selected the codon-optimized xylanase gene AnXlnB, the β-xylanase gene AnXlnD, and the acetylxylanase gene CcXynA. Saccharomyces cerevisiae strains with the decomposition and utilization capacity of pentosan were obtained. The ability of pentosan fermentation to produce alcohol was tested in a specific medium.(Figure 2).

Experimental approach

1. Aspergillus niger derived xylanase and β-xylosidase expression plasmid construction For the effective utilization of the xylan component present in the wheat B starch, the Aspergillus niger derived β-xylosidase (Figure 4A) was cloned together with the xylanase expression gene. To make the final plasmid pXylan-BD (Figure 4B), the pXlnD plasmid was constructed firstly. The promoter TPI1, codon-optimized AnXlnD CDS, and CYC1 terminator PCR bands were shown in the Figure 3A, 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 3A lane 7 and Figure 3B 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 3C) was also cut with the same enzyme, these three parts were ligated to make the final plasmid pXylan-BD.

Figure 2. 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 5 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.

2. sequence information of the final plasmids

Figure 3. Figure 3: Plasmids DNA sequencing. A: pXlnB plasmid. B: pXlnD plasmid..

The blast result shows that the plasmid is constructed successfully.

Figure 4. Positive colonies verification through the colony PCR.A: the results of colony PCR, B: DNA sequencing of the plasmid pXylan-B, C: and pXylan-BD..

Figure 7A 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 7B and 7C showed that both the pXylan-B and pXylan-BD plasmids were constructed successfully.

Proof of function

1. fermentation test

Figure 5. 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 undergo 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 8B, 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.

References

1. 王良东. 小麦B淀粉的组分, 性质和利用的研究[D]. 江南大学, 2004.

2. 赵银峰. 小麦酒精发酵新工艺的研究[D]. 郑州大学, 2005.

3. Claes A, Deparis Q, Foulquié-Moreno M R, et al. Simultaneous secretion of seven lignocellulolytic enzymes by an industrial second-generation yeast strain enables efficient ethanol production from multiple polymeric substrates[J]. Metabolic engineering, 2020, 59: 131-141.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 190
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 283
  
• 1000
  COMPATIBLE WITH RFC[1000]