Part:BBa_K3022002

ppk1 in Citrobacter freundii ATCC 8090

Natural function of part: The polyphosphate kinase in Citrobacter freundii ATCC 8090 is responsible for its intracellular inorganic polyphosphate (polyP) production via reversibly catalyzing the transfer of terminal phosphate from ATP to a growing polyP chain.

Wild-type Citrobacter freundii ATCC 8090 was purchased from China Center of Industrial Culture Collection (CICC, China). The ppk1 gene was acquired by PCR. This organism is our chasiss, in which its native PPK1 will be overexpressed with a plasmid of medium-copy numbers.

Although PPKs from E. coli and C. freundii shares 96% amino acids’ identity, the C. freundii PPK has a glutamate and a lysine residue in positions 327 and 328, where in E. coli they are substituted by much less strongly charged alanine and glutamine residues, respectively. Although these natural occurred mutation sites found in C. freundii PPK are distant from the enzymes’ active site, they lie in the interfaces among monomers of the PPK tetramer. Benefit from this difference, a dramatic increase of intracellular polyP accumulation can be achieved with C. freundii.

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]

Experimental characterization added by CPU-Nanjing 2022 TEAM

Team CPU-Nanjing has added new experimental characterization on this existing Part.

Polyphosphate (PolyP) is a polyanionic biopolymer and generally stored in cells in the form of polyP granules. The presence of intracellular polyP granules can be observed with light microscopy after the cells had been stained with the cationic dye, such as toluidine blue. To test this, we overexpressed this Part using plasmid pBBR1MCS2 in E. coli K12. When phosphate was used as the phosphorus source, the engineered strain can produce intracellular polyP granules.

Figure 1. PolyP granules in E. coli K12/CFPPK. Intracellular polyP granules appear blue-purple to blue-black. Scale bar, 5 μm.

After staining, polyP can be easily visualized as blue-purple granules.

Improvement made by CPU-Nanjing 2022 TEAM

CPU-Nanjing has improved the existing part by coupling it with the phosphite dehydrogenase from Klebsiella pneumonia (KPD, BBa_K2325001), and the number of the improved part is BBa_K4257033.

Given that phosphite is not the form of phosphorus available to life and even toxic to life in high concentration, the bacterium cannot use phosphite to grow except that it harbors phosphite dehydrogenase. The polyphosphate kinase of Citrobacter freundii ATCC 8090 (CFPPK) catalyzes the synthesis of polyphosphate (polyP) using ATP as the substrate, in which the phosphorus is essentially derived from exogenous phosphate rather than phosphite. The KPD catalyzes the oxidation of phosphite to phosphate. Therefore, the combination of KPD and CFPPK can confer the host bacterium with a new capacity that synthesis polyP directly from phosphite. More importantly, given most bacteria cannot use phosphite, the engineered strain harboring our improved Part can produce polyP under “unsterile” condition. We validated the function of KPD+CFPPK by performing intracellular polyP synthesis using phosphite as the substrate.

We constructed two engineered E. coli K12 strains that overexpress CFPPK and KPD+CFPPK, respectively. Nutrient-poor synthetic municipal wastewater (SMW) medium with phosphite (P, +3 valance) as the solo phosphorus source was adopted to evaluate polyP synthesis.

1. Cell growth

The engineered E. coli K12 strains were inoculated into the SMW (P, +3 valance) to attain an initial OD600 of 0.05. After cultivation, bacterial cell growth was determined by optical density measurements.

Figure 1. Maximum optical density of each engineered E. coli K12 strain grown in SMW (P, +3 valance).

Only K12/KPD+CFPPK can utilize phosphite to grow, whereas K12/CFPPK cannot (Figure 1).

2. PolyP synthesis

Intracellular polyP assay showed that, associated with the growth of K12/KPD+CFPPK is its intracellular polyP synthesis (Figure 2). By contrast, no polyP was detected in the cells of K12/CFPPK.

Figure 2. Intracellular polyP content. N.D., not detected.

3. Microscopic observation

Consistent with quantitative polyP assay, polyP granules were only observed in the cells of K12/KPD+CFPPK (Figure 3).

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

iGEM2021_Nanjing-China Experiment

Group: Nanjing-China 2021
Author: Hao Yin

We took ppk1 in Citrobacter freundii ATCC 8090 as a comparison and validated its activity through the experiment. We found that like in Citrobacter freundii ATCC 8090, ppk1 can also produce polyP in the E.coli, which proved our hypothesis.After incubation in the polyphosphorous culture medium for 20 min, we found that polyP was produced by PPK1, which was proved by ashing and spectrum.

We also measured the curve of OD value CPVM in PA medium. Here is our result:

Figure 1）OD measurement of CPVM

We compared results of CPVM with results of EPVM, and proved the activity of ppk1 in Citrobacter freundii ATCC 8090.

iGEM2020_Nanjing-China Experiment

Group: Nanjing-China 2020
Author: Guangyu Hu

We found that polyP productivity would reach the highest after inoculation for 14 hours and 100ml of synthetic wastewater in a 200ml Erlenmeyer flask has the highest output and the highest conversion efficiency.

PolyP formation is closely associated with ppk1 gene in CPP. This means the cultivation time and volume will affect P consuming a lot. After comparing different cultivation conditions, we found that polyP productivity will reach the highest after inoculation for 14 hours, that is approximately 20mg/L. Besides, 100ml of synthetic wastewater in a 200ml Erlenmeyer flask has the highest output and the highest conversion efficiency. Without bacterial contamination and normal bacterial viability，it can almost convert all inorganic phosphates into polyphosphates.

iGEM2019_Nanjing China Experiment

This year our team develops a simple solo medium-copy plasmid-based polyphosphate kinase (PPK1) overexpression strategy for achieving maximum intracellular polyphosphate accumulation.

We test plasmid-borne ppk1 expression in CPP via qRT-PCR analysis, supernatant Pi concentration and optical density of CPP and CWT in Synthetic municipal wastewater(SMW).

Ps: SMW means Synthetic municipal wastewater

CPP means solo medium-copy C. f reundii ATCC8090 ppk in C. f reundii ATCC8090

CWT means wild type C. f reundii ATCC8090

Figure 1）qRT-PCR analysis of plasmid-borne ppk1 expression in CPP

Figure 2）Pi absorption&release

Figure 3）Optical density of CPP grown in SMW compared with CWT

Reference: Wang X , Wang X , Hui K , et al. Highly Effective Polyphosphate Synthesis, Phosphate Removal and Concentration Using Engineered Environmental Bacteria Based on a Simple Solo Medium-copy Plasmid Strategy[J]. Environmental Science & Technology, 2017:acs.est.7b04532.