Difference between revisions of "Part:BBa K3996015"

Revision as of 05:41, 20 October 2021

pXylan

pXylan

Profile

Name: pXylan

Base Pairs: 7473bp

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.

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

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.

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

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