Part:BBa_K4257033

KPD+CFPPK

KPD (BBa_K2325001) is the native phosphite dehydrogenase from Klebsiella pneumonia (Relyea and Van Der Donk 2005), whereas CFPPK (BBa_K3022002) is the native polyphosphate kinase from Citrobacter freundii ATCC 8090 (Wang et al. 2018). Both Parts are randomly picked from the documented Basic Part. This year, our team developed a new metabolic pathway by coupling RPD (BBa_K4257011, native phosphite dehydrogenase from Ralstonia sp.) and PPK-M (BBa_K4257000, a mutant E. coli polyphosphate kinase). This pathway conferred E. coli K12 with the capacity of phosphite oxidation and polyphosphate synthesis. As such, the engineered E. coli K12 can produce phosphate with phosphite as raw material. To confirm that our strategy has the general applicability without being confined to the enzymes we used, we composited this new Part, KPD+CFPPK, and tested it.

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 test of KPD+CFPPK was performed using the same vector and host cell. Both KPD and CFPPK served as the control. E. coli K12 harboring pBBR1MCS2/KPD+CFPPK was designated MKCP, whereas E. coli K12 harboring pBBR1MCS2/KPD or pBBR1MCS2/CFPPK was designated MK and MCP, respectively.

1. Phosphite utility test

Phosphite utility test was performed in synthetic municipal wastewater medium (Wang et al. 2018) with phosphite (P, +3 valence) as the solo phosphorus source.

Figure 1. Supernatant phosphite assay.

When grown in SMW (P, +3 valence), MKCP and MK can use phosphite to grow, whereas MCP cannot. Therefore, the MCP culture showed no decrease in supernatant phosphite (Figure 1).

2. PolyP accumulation

MKCP consumed more phosphite as compared with MK (Figure 1). The extra portion of phosphorus consumed by MKCP are stored in MKCP in the form of intracellular polyP (Figure 2), thereby resulting in a high phosphorous content for MKCP (Figure 3).

Figure 2. Light microscopy images of stained cells. PolyP granules appear blue-purple to blue-black. Scale bar, 5 μm.

Figure 3. Cellular phosphorus content determination.

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 MKCP.

Figure 4. Supernatant phosphate assay.

Although the final phosphate yield may vary with different combinations of the Basic Parts, the above results were sufficient enough to confirm the general applicability of our strategy.

References

Relyea, H.A. and Van Der Donk, W.A. (2005) Mechanism and applications of phosphite dehydrogenase. Journal of Biological Chemistry (3), 171-189.

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.