Part:BBa_K912001

E. coli phytase (appa phytase)

Part to produce the appa e. coli phytase. This phytase acts to break down phytic acid into inositol, releasing chelated iron and other micronutrients.

HokkaidoU_Japan 2017 analyzed this part. This protein's activity falls as below graph.

Protocol: 50μl 7.5 mM Phytic acid 190μl CH3COOH Buffer

Digest phytic acid in above situation with sample phytase solution 10μl ,37degrees celcius, 10 min.

After digestion, using AMM solution (2ml of 10mM NH4Mo7O2.4H2O: 5N H2SO4: Acetone= 1:1:2) analyze Optical Absorbance. The more Pi are there, the more yellow AMM becomes.

Contribution by 2023 iGEM Team Fujian-United

Summary

Figure 1 Expression frame of the AppA display plasmid

We have made contributions on the original components BBa_K912001. Throughout the design, we used GAL1 promoters (BBa_K4870008) to induce protein expression using galactose and added MF-alpha-1(BBa_K4870009), which is N-terminal secretion signal from S. cerevisiae alpha-factor, and connected SED1 gene (BBa_K4870011) through G4S 3-linker (BBa_K4870010). Fusion expression of phytase encoding genes AppA with anchoring protein encoding genes SED1 on the cell surface to produce yeast single cell proteins with phytase activity, which can be added to feed to enhance animal nutrition and absorption.

Documentation

Usage and Biology

Appa（appa phytase）acts to break down phytic acid into inositol, releasing chelated iron and other micronutrients. Phytase belongs to the histidine or alkaline phosphatase family and can catalyze the hydrolysis of phytic acid to gradually produce inositol derivatives and inorganic phosphates. Therefore, phytase is usually used as a commercial feed additive to improve the digestibility of phytic acid in grain raw materials. On the one hand, it increases the animal's absorption of phosphate in feed, and on the other hand, it reduces the anti nutritional effect of phytic acid[1-3].

Characterization/Measurement

To construct the plasmid pYES2-Hyg-GAL1p-α-AppA-SED1-RPL41Bt, we used the E. coli DH5α genomic DNA as a template to amplify the AppA sequence by PCR. Then, the AppA sequence was inserted into the SphⅠ and AvrⅡ sites of plasmid pYES2-Hyg-GAL1p-α-SED1-RPL41Bt, by restriction endonuclease digestion and linkage.

The plasmid was then transferred into the E. coli DH5α competent cells. The result showed that transformants were successfully grown after overnight culture. After colony PCR verification, the recombinants had the expected bands (1293 bp), indicating the successful transformation of this plasmid.

Figure 2: Construction of the AppA display plasmid

References

1. Dersjant-Li Y, Bello A, Stormink T, Abdollahi MR, Ravindran V, Babatunde OO, Adeola O, Toghyani M, Liu SY, Selle PH, Marchal L. Modeling improvements in ileal digestible amino acids by a novel consensus bacterial 6-phytase variant in broilers. Poult Sci. 2022 Mar;101(3):101666.
2. Acquistapace IM, Thompson EJ, Kühn I, Bedford MR, Brearley CA, Hemmings AM. Insights to the Structural Basis for the Stereospecificity of the Escherichia coli Phytase, AppA. Int J Mol Sci. 2022 Jun 6;23(11):6346.
3. Navone L, Vogl T, Luangthongkam P, Blinco JA, Luna-Flores C, Chen X, von Hellens J, Speight R. Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris. Microb Cell Fact. 2021 Jan 7;20(1):8. doi: 10.1186/s12934-020-01499-7.

Improvement: AppA Phytase (BBa_K912001)

Summary

In this project, to expand the application of AppA phytase (BBa_K912001) in feed, we inserted the AppA gene into the plasmid pYES2-Hyg-GAL1p-α-SED1-RPL41Bt (BBa_K4870016) and transformed it into yeast cells. Additionally, we utilized yeast cell surface display technology and supplemented the characterization of AppA phytase activity displayed on yeast under different pH and temperature conditions. For the reaction temperature, the activity of this phytase increased with rising temperature, with the highest activities detected at 55 °C.

Documentation

a. Usage and Biology

We transformed the plasmid into yeast and fused the phytase encoding gene with the anchoring protein encoding gene on the cell surface to produce yeast single cell protein with phytase activity, which can be added to feed to enhance animal nutrition and absorption[1],[2].

Figure 1: Design diagram of this project. Image by LIU JINLE.

b. Characterization/Measurement

The positive transformant was inoculated, and plasmid was extracted using a commercial miniprep kit. Before the transformation, the BY4741 strain was inoculated and prepared in a competent state. Then, the lithium acetate (LiAc) transformation method was used to transform linearized plasmid into BY4741 competent cells, followed by incubation at 30 °C for 48 h. The colony PCR identification confirmed that the plasmid was successfully transfected into BY4741 cells. Sequencing of the transformants verified the correct sequence of the AppA target fragment.

Figure 2: Results of AppA display plasmid transformation of yeast cells.

We selected a positive transformant and cultured it to the logarithmic growth period. Galactose was then added to induce the promoter, and yeast cells displaying phytase on the surface were obtained. Cells were pelleted by centrifugation and washed by resuspension in 50 mM sodium acetate buffer (pH 5.0) A portion of the suspension was sonicated and then centrifuged to obtain lysate supernatant and precipitate containing cell wall-bound phytase. The SDS-PAGE result showed that the AppA was successfully displayed on the yeast cell surface (Figure 3).

Figure 3: AppA SDS-PAGE and phytase activity under different (A) pH and (B) temperature conditions.

References

1. Chen X, Xiao Y, Shen W, Govender A, Zhang L, Fan Y, Wang Z. Display of phytase on the cell surface of Saccharomyces cerevisiae to degrade phytate phosphorus and improve bioethanol production. Appl Microbiol Biotechnol. 2016 Mar;100(5):2449-58. doi: 10.1007/s00253-015-7170-4.
2. Navone L, Vogl T, Luangthongkam P, Blinco JA, Luna-Flores C, Chen X, von Hellens J, Speight R. Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris. Microb Cell Fact. 2021 Jan 7;20(1):8.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 688
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1213