Part:BBa_K3996007

pOdd-1

pOdd-1

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 reconstructed the plasmid pAR318 by PCR for further use.

Figure 2. DNA sequence map of plasmid pOdd-1..

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.

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. 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

2. Srinivasan, P., Smolke, C.D. Engineering a microbial biosynthesis platform for de novo production of tropane alkaloids. Nat Commun 10, 3634 (2019).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1706
  Illegal BsaI.rc site found at 1694
  Illegal SapI site found at 1672
  Illegal SapI.rc site found at 1727