Part:BBa_K3996013
pXylan-B
pXylan-B
Profile
Name: pXylan-B
Base Pairs: 1593bp
Origin: Saccharomyces cerevisiae, synthesis
Properties: pentosan fermentation to produce alcohol
Usage and Biology
Wheat B starch is a by-product of wheat starch deep processing, which is often directly used as feed, with low industrial added value. If wheat B starch is used as raw material to produce alcohol, part of the shortcomings of wheat starch alcohol can be avoided and the utilization value of wheat B starch can be improved. After sugar production of wheat B starch by liquid saccharification pretreatment, it can use conventional brewing yeast to produce alcohol, but this process composition of pentosan in wheat B starch did not use, even in the pretreatment stage to join pentosan enzyme, xylose and arabinose (pentose monosaccharides will use by conventional saccharomyces cerevisiae, at the same time the extra pentosan enzyme also increases the cost of production. Therefore, it is ideal to develop saccharomyces cerevisiae strains with the ability of autocrine pentosanase and pentose utilization.
Construct design
The plasmid is engineered for further use. (Figure 2).
The profiles of every basic part are as follows:
BBa_K3996000
Name: GAP promoter Base Pairs: 667bp Origin: Saccharomyces cerevisiae, genome Properties: A constitutive expression promoter
Usage and Biology
The glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) has been used for constitutive expression of many heterologous proteins. The pGAP-based expression system is more suitable for large-scale production because the hazard and cost associated with the storage and delivery of large volume of methanol are eliminated.
BBa_K3996003
Name: CYC1 Base Pairs: 250bp Origin: Saccharomyces cerevisiae, genome Properties: Common transcriptional terminator
Usage and Biology
This is a common transcriptional terminator. Placed after a gene, it completing the transcription process and impacting mRNA half-life. This terminator can be used for in vivo systems,and can be used for modulating gene expression in yeast.
BBa_K3996004
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.
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).
(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 3A, 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 3A lane 4 and Figure 3B 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 3C) was also cut with the same enzyme, these two parts were ligated to make the final plasmid pXylan-B. 2. Verification of the plasmids via colony PCR
From the Figure 4, we can figure out that the numbers 10, 12, and 14 were the positive colonies of the plasmid pXylan-B, and Number 12 was sent for sequencing.
Proof of function
1. fermentation test
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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