Part:BBa_K3996014

pXylan-BD

pXylan-BD

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.

Figure 1. Principle diagram of pentosan fermentation..

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).

Figure 2. DNA map of plasmid pXlnB and pXylan-B..

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).

Figure 3.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 4. 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 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. 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.

3. sequence information of the final plasmids

Figure 6. Plasmids DNA sequencing. A: pXlnB plasmid. B: pXlnD plasmid..

The blast result shows that the plasmid is constructed successfully.

Figure 7. 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 8. 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.

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.

Figure 9. The blast results about the DNA sequence of our new part BBa_K3996014 and the old parts BBa_K2365052..

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

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 2369
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2462
  
• 1000
  COMPATIBLE WITH RFC[1000]