Difference between revisions of "Part:BBa K3996004"

Latest revision as of 03:31, 20 October 2021

AnXlnB orf

AnXlnB orf

Profile

Name: AnXlnB orf

Base Pairs: 678bp

Origin: synthesis

Properties: Endohydrolysis of (1->4)-beta-D-xylosidic linkages in xylans

Usage and Biology

This protein is involved in the pathway xylan degradation, which is part of Glycan degradation. Endo-1,4-beta-xylanase 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

The plasmid is engineered for further use. (Figure 1).

Figure 1. DNA map of plasmid pXylan-B..

Experimental approach

1. Fragments PCR products Electrophoresis 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 2).

Figure 2. Plasmids construction used fragments PCR amplification..

Lane 2: AnXlnB CDS, 706 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.

Proof of function

1. fermentation test

Figure 3. 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