Part:BBa_K4002006

HR-L-endo-pgaA-HR-R

HR-L-endo-pgaA-HR-R

Profile

Name: HR-L-endo-pgaA-HR-R

Base Pairs: 2135 bp

Origin: Ssynthetic

Properties: CRISPR technology repair template for build a type of multi-functional yeast

Usage and Biology

Polygalacturonase is an enzyme that hydrolyzes the alpha-1,4 glycosidic bonds between galacturonic acid residues. It is also known as pectin depolymerase, PG, pectolase, pectin hydrolase, and poly-alpha-1,4-galacturonide glycanohydrolase. Pectin is a significant carbohydrate component that comprises plant cell walls. The brewer's yeast uses sugar in the fruit juice to produce alcohol, and pectinase destroys the pectin located in the cell wall to improve the juice yield and eliminate the cloudiness of the fruit wine. HR-L and HR-R are homology arm, refers to the flanking sequence on both sides of the HXK1.

Construct design

HR-L is the homology arm upstream HXK1. HR-R is the homology arm downstream HXK1. pgaA is the sequence of pgaA inserted in the homology arm (Figure 1 and 2).

Figure 1. HR-L-endo-pgaA-HR-R box..

Figure 2. Schematic map of HR-L-endo-pgaA-HR-R..

The profiles of every basic part are as follows:

BBa_K4002000

Name: HR-L

Base Pairs: 500bp

Origin: Saccharomyces cerevisiae, genome

Properties: A coding sequence of left homology arm

Usage and Biology

This is a coding sequence of left homology arm, refers to the flanking sequence on one side of the HXK1 sequence, which is completely consistent with the genome sequence, and is used to identify and recombine the region. system (T6SS).

BBa_K4002001

Name: HR-R Base Pairs: 522bp Origin: Saccharomyces cerevisiae, genome Properties: A coding sequence of right homology arm

Usage and Biology

This is a coding sequence of right homology arm, refers to the flanking sequence on one side of the HXK1 sequence, which is completely consistent with the genome sequence, and is used to identify and recombine the region.

BBa_K4002005

Name: endo-pgaA Base Pairs: 1113bp Origin: Aspergillus niger SC323, genome Properties: An enzyme degradation of pectin

Usage and Biology

Polygalacturonase is an enzyme that hydrolyzes the alpha-1,4 glycosidic bonds between galacturonic acid residues. It is also known as pectin depolymerase, PG, pectolase, pectin hydrolase, and poly-alpha-1,4-galacturonide glycanohydrolase.

Experimental approach

1. Construction of repair template The repair template DNA containing PgaA gene (Fig. 3A) was generated by the overlap-PCR method. Firstly, the DNA fragments of upstream and downstream homologous regions were amplified with ~20 bp ends overlapping to the PgaA gene, producing ~500 bp PCR products (Fig. 3B). Secondly, the two fragments were annealed to the 5’- and 3’-ends of PgaA. Finally, the annealed products were further amplified using end primers of HR-L and HR-R, which resulted in a fragment of 1.5 kbas verified by agarose gel electrophoresis and DNA sequencing (Figs. 3C and 3D).

Figure 3. Construction of repair template. (A) Schematic representation of repair template; (B) Agarose gel electrophoresis of PCR products; (C) DNA sequencing result analysis..

2. Yeast strain transformation and positive transformants verification The constructed CRISPR plasmids and repair template DNA were chemically transformed into the S. cerevisiae strains. The positive transformants were selected against YPD medium supplemented with Nours and hygromycin. The resulting colonies were picked up and cultured. To investigate whether the PgaA gene was integrated into yeast genome, we performed PCR experiments using the upstream and downstream primers complementary to HR-L and HR-R genes, respectively. As shown in Fig. 4A, we obtained specific PCR products with expected size of ~1500 bp. The DNA fragments were then extracted and purified for sequencing. The sequencing results finally confirmed that the PgaA gene was successfully integrated into S. cerevisiae genome (Fig. 4B).

Figure 4. Verification of PgaA containing transformants. (A) Agarose gel electrophoresis of PCR products; (B) DNA sequencing result analysis..

Proof of function

Pectinase activity assay The pectinase activities of PgaA were determined using the dinitrosalicylic acid (DNS) colorimetric method. Briefly, in the presence of PgaA, pectin can be degraded into galacturonic acids, which reacts with DNS to form a compound with a maximum absorption at 540 nm. Thus, the activity of PgaA can be calculated by measuring the absorbance of the reactants with a spectrophotometer. For accurate quantification, a standard curve was generated using a series of concentrations of pectinase standards. As shown in Table. 2 and Fig. 5, the concentration of enzyme correlates well with the absorbance detected at 540 nm, applying to the Lambert-Beer law.

Table 2. Measurement of standard pectinase activities at different concentration.

Figure 5. Standard curve of pectinase.

With this standard curve, we next determined the concentration of PgaA from recombinant S. cerevisiae strains. Samples from the culture media, total cell lysates and the soluble portion of cell lysates were collected and subjected to DNS colorimetric assay. As shown in Table. 3, the concentration of PgaA in the culture media of sample -1 and -2 were determined at about 0.034 mg/ml and 0.028 mg/ml, respectively, which were relatively higher than that of cell lysates (0.009 mg/ml and 0.007 mg/ml), suggesting that most of the PgaA proteins were secreted into the culture media. In addition, in the cell lysates of sample 1, we detected ~76% of PgaA in the soluble supernatants, implying that most of the PgaA in cells are soluble. Unexpectedly, the concentration of PgaA in the soluble supernatants of sample 2 was higher than that of total cell lysates, this could be due to experimental mistakes.

Table 3. Measurement of PgaA concentration and unit of activity in various samples.

We successfully prepared genetically engineered wine yeast strain which contains pectinase in its genome. The pectinase produced from yeast well degrade pectin into small sugars.

References

1.Nishimasu, H., et al. Cell. 2014

2.James E.D., et al. Nucleic Acids Res. 2013

3.https://www.asme.org/topics-resources/content/8-ways-crisprcas9-can-change-world

4.https://www.nature.com/articles/s41599-019-0319-5

5.https://www.sciencedirect.com/topics/food-science/fruit-wine

6.崔凯宇,李迎秋.果胶酶生产和应用的研究进展[J].江苏调味副食品,2016(01):11-13.

7.Yang J , Luo H , Jiang L , et al. Cloning, expression and characterization of an acidic endo-polygalacturonase from Bispora sp. MEY-1 and its potential application in juice clarification[J]. Process Biochemistry, 2011, 46(1):272-277.

8.李烨青. 真菌来源的嗜热果胶酶基因挖掘及其催化效率的改造[D].江西农业大学,2017.

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 931
  Illegal BglII site found at 1489
  Illegal XhoI site found at 1449
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1188
  Illegal BsaI.rc site found at 690
  Illegal BsaI.rc site found at 1560