Part:BBa_K3996014
pXylan-BD
pXylan-BD
Profile
Name: pXylan-BD
Base Pairs: 4842 bp
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
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).
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_K3996001
Name: TPI1 promoter
Base Pairs: 586bp
Origin: Saccharomyces cerevisiae, genome
Properties: A constitutive expression promoter
Usage and Biology
Triose phosphate isomerase 1 promoter (TPI1 promoter) is used for regulating gene expression in yeast.
BBa_K3996002
Name: FBA1 promoter
Base Pairs: 586bp
Origin: Saccharomyces cerevisiae, genome
Properties: A constitutive expression promoter
Usage and Biology
The FBA1 promoter activity was 2.2 and 5.5 times stronger than the TDH1 and GPM1 promoters, respectively.
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.
BBa_K3996005
Name: AnXlnD orf
Base Pairs: 2415bp
Origin: synthesis
Properties: Hydrolysis of (1->4)-beta-D-xylans, to remove successive D-xylose residues from the non-reducing termini
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.
BBa_K3996006
Name: CcXynA orf
Base Pairs: 1563bp
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.
BBa_K3996007
Name: pOdd-1
Base Pairs: 1770bp
Origin: synthesis
Properties: A Yeast Expression plasmids backbone
Usage and Biology
Reconstructed by PCR based on plasmid pAR318.
BBa_K3996008
Name: pOdd-2
Base Pairs: 1770bp
Origin: synthesis
Properties: A Yeast Expression plasmids backbone
Usage and Biology
Reconstructed by PCR based on plasmid pAR318.
BBa_K3996009
Name: pOdd-3
Base Pairs: 1770bp
Origin: synthesis
Properties: A Yeast Expression plasmids backbone
Usage and Biology
Reconstructed by PCR based on plasmid pAR318.
BBa_K3996010
Name: pXlnB
Base Pairs: 1593bp
Origin: synthesis
Properties: A Yeast Expression plasmids backbone
Usage and Biology
It is formed with pOdd-1 plasmid backbone, GAP promoter, AnXlnB orf and CYC1 terminator.
BBa_K3996011
Name: pXlnD
Base Pairs: 3249bp
Origin: synthesis
Properties: A Yeast Expression plasmids backbone
Usage and Biology
It is formed with pOdd-2 plasmid backbone, TPI1 promoter, AnXlnD orf and CYC1 terminator.
BBa_K3996012
Name: pXynA
Base Pairs: 2631bp
Origin: synthesis
Properties: A Yeast Expression plasmids backbone
Usage and Biology
It is formed with pOdd-3 plasmid backbone, FBA1 promoter, AnXlnD orf and CYC1 terminator.
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).
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
For the effective utilization of the xylan component present in the wheat B starch, the Aspergillus niger derived β-xylosidase (Figure 4A) was also 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 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.
3. sequence information of the final plasmids
The blast result shows that the plasmid is constructed successfully.
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
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.
Improvement of an existing part
Compared to the old part BBa_K2365052, set up a TPI1 promoter-CYC1 terminator for gene expression regulation in yeast, we design a new part BBa_K3996014, which contains the TPI1 promoter-CYC1 terminator and a new enzyme AnXlnB. The AnXlnB protein is involved in the pathway xylan degradation.
The group iGEM17_NAU-CHINA aimed to use reserved restriction enzyme cutting site to inserted GPF (S6GT) between promoter and termiator as the index of promoter test in S. cerevisiae. As a result, they detected the GFP regulated by different promoters.
Based on the these groups’ contribution, our team design the new composite part BBa_K3996014 to express AnXlnB. After the composite part was inserted in a particular plasmid vector and transformed into BY4741. The different regulatory properties in the fermentation are measured, so as to achieve the purpose of our project. First of all, we constructed a composite part BBa_K3996013 which contains several regulatory elements and transformed it into S. cerevisiae. Furthermore, in order to have a general idea of fermentation, we measured the OD600 and the concentration of sugar in the culture, we found that 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]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2369
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2462
- 1000COMPATIBLE WITH RFC[1000]
None |