Part:BBa_K3996015

pXylan

pXylan

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

The plasmid is engineered for further use. (Figure 2)

Figure 2. DNA map of plasmid pXylan..

The profiles of every basic part are as follows:

BBa_K3996000

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

Usage and Biology

Triose phosphate isomerase 1 promoter (TPI1 promoter) is used for regulating gene expression in yeast.

BBa_K3996002

Usage and Biology

The FBA1 promoter activity was 2.2 and 5.5 times stronger than the TDH1 and GPM1 promoters, respectively.

BBa_K3996003

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

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

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

Usage and Biology

This protein is involved in the pathway xylan degradation, which is part of Glycan degradation.

Experimental approach

Fragments PCR products Electrophoresis

To utilize the xylan component contained in the wheat B starch, we cloned the xylanase expression gene from Aspergillus niger.

Figure 3. Plasmids construction used fragments PCR amplification..

Lane 2: pXlnD plasmid backbone fragment, 1804 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.

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
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 5727
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 5727
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2369
  Illegal BglII site found at 6120
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 5727
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 5727
  Illegal NgoMIV site found at 6904
  Illegal AgeI site found at 2462
  Illegal AgeI site found at 6497
  
• 1000
  COMPATIBLE WITH RFC[1000]