Part:BBa_K4257022

RPD+PPK-M

RPD (BBa_K4257011) is a phosphite dehydrogenase, which catalyzes the oxidation of phosphite to phosphate (Hirota et al. 2012). Given that phosphite is not the form of phosphorus available to life and even toxic to life in high concentration, bacteria cannot use phosphite to grow. PPK-M is (BBa_K4257000) a mutant of E. coli polyphosphate kinase, which catalyzes the synthesis of polyphosphate (polyP) using ATP as the substrate (Rudat et al. 2018). In host cells, regeneration of intracellular ATP necessitated the use of phosphate. Generally, intracellular polyP is synthesized under aerobic conditions and will be released back to into ambient environment in the form of phosphate under anaerobic conditions. Therefore, co-expression of RPD and PPK-M would construct a new metabolic pathway, in which phosphite is the raw substrate, polyP is the intermediate, and phosphate is the final product.

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
  COMPATIBLE WITH RFC[1000]

Data:CPU-Nanjing 2022 TEAM

The function of RPD+PPK-M was identified using pBBR1MCS2 as the vector and E. coli K12 as the host cell. Both RPD and PPK-M served as the control. E. coli K12 harboring pBBR1MCS2/RPD+PPK-M was designated MRP, whereas E. coli K12 harboring pBBR1MCS2/RPD or pBBR1MCS2/ PPK-M was designated MR and MP, respectively.

1. Phosphite (P, +3 valence) utility test

Phosphite utility test was performed in synthetic municipal wastewater (SMW, a nutrient-poor synthetic medium) with phosphite as the solo phosphorus source (Wang et al. 2018).

Figure 1. Supernatant phosphite assay.

When grown in SMW (P, +3 valence), MRP and MR can use phosphite, as evidenced by the decrease in supernatant phosphite (Figure 1).

2. PolyP accumulation

As shown in Figure 1, MRP consumed more phosphite as compared with MR and this resulted in a high phosphorous content for MRP (Figure 2).

Figure 2. Cellular phosphorus content determination.

Toluidine blue staining showed that the extra portion of phosphorus are stored in MRP cells in the form of intermediate polyP (Figure 3).

Figure 3. Light microscopy images of stained cells. PolyP granules appear blue-purple to blue-black. Scale bar, 5 μm.

3. Phosphate as final product

After ten-fold concentration of each culture, they were subjected to anaerobic phosphate release. High concentration of phosphate was only detected in the supernatant of concentrated MRP (Figure 4). This result confirmed the feasibility of our strategy based on this new composite Part.

Figure 4. Supernatant phosphate assay.

Although MR can utilize a certain amount of phosphite, it can not be used to produce phosphate (Figures 1 and 4). This is because the phosphite it oxidized was just incorporated into its cell components to meet its own growth needs.

References

Hirota, R., Yamane, S.-t., Fujibuchi, T., Motomura, K., Ishida, T., Ikeda, T. and Kuroda, A. (2012) Isolation and characterization of a soluble and thermostable phosphite dehydrogenase from Ralstonia sp. strain 4506. Journal of Bioscience Bioengineering 113(4), 445-450.

Rudat, A.K., Pokhrel, A., Green, T.J. and Gray, M. (2018) Mutations in Escherichia coli polyphosphate kinase that lead to dramatically increased in vivo polyphosphate levels. Journal of Bacteriology 200(6), e00697-00617.

Wang, X., Wang, X., Hui, K., Wei, W., Zhang, W., Miao, A., Xiao, L. and Yang, L. (2018) Highly effective polyphosphate synthesis, phosphate removal, and concentration using engineered environmental bacteria based on a simple solo medium-copy plasmid strategy. Environmental Science Technology 52(1), 214-222.