| CPU_Nanjing - iGEM 2022

Part 1 Introduction of phosphate manufacture pathway by generating gene-cassette by coupling phosphite oxidation and polyphosphate synthesis

Molecular level

Following standard molecular cloning procedure, we obtain several plasmids and corresponding E. coli K12 derivatives. Detailed information on these DNA constructs and the derivative strains are listed in Table 1.

Table 1 Detailed information on DNA constructs and derivative strains.
Plasmids and Strains	Description
pBBR1MCS2/RPD	pBBR1MCS2 with phosphite dehydrogenase gene (RPD) [1]
pBBR1MCS2/PPK-M	pBBR1MCS2 with mutant polyphosphate kinase gene (PPK-M) [2]
pBBR1MCS2/RPD+PPK-M	pBBR1MCS2 with RPD+PPK-M
MR	E. coli K12 harboring plasmid pBBR1MCS2/RPD
MP	E. coli K12 harboring plasmid pBBR1MCS2/PPK-M
MRP	E. coli K12 harboring plasmid pBBR1MCS2/RPD+PPK-M

All DNA constructs were confirmed by sequencing. After electroporation, derivative strains were obtained by selecting from LB agar plates containing 50 mg/L kanamycin (Figure 1).

Figure 1. Positive colony grown on LB agar plates containing 50 mg/L kanamycin.

Transcriptional level

Given that intracellular ATP is involved in the engineered metabolic pathway, we adopted medium-copy pBBR1MCS2 to overexpress the target gene [3], alleviating the metabolic burden imposed upon the host cell. Under such circumstances, transcriptional analysis will be a more sensitive method to detect plasmid-borne gene expression.

qRT-PCR analysis confirmed that both RPD and PPK-M are successfully overexpressed, though the expression level of PPK-M showed a slight decrease when it was expressed from the RPD+PPK-M-cassette.

Figure 2. qRT-PCR analysis of RPD and PPK-M in E. coli K12 derivatives sampled from synthetic
municipal wastewater (SMW) medium. Results are presented relative to the average expression level of 16s rDNA gene, set as 1.

Part 2 Identification of intracellular intermediate and extracellular final product

2.1 Phosphite utilization test

When cultured in synthetic municipal wastewater (SMW, a nutrient-poor synthetic medium [4, 5] that mimicking the situation that may occurred on terrestrial planets) with phosphite (P, +3 valence) as the solo phosphorus source, only MR and MRP can grow whereas WT and MP cannot (Figure 3).

Figure 3. Optical density of each strain grown in SMW (P, +3 valence).

Supernatant and intracellular phosphorus measurements showed that the growth of MR and MRP in SMW was attributed to their capability to uptake exogenous phosphite (P, +3 valence) and oxidize it to assimilable phosphate (P, +5 valence; Figures 4).

Figure 4. Supernatant phosphite concentration of each strain
grown in SMW (P, +3 valence), expressed as milligrams of phosphorus per liter.

Although MR possesses the capacity of phosphite oxidation (Figure 4), it does so just to meet its own growth needs (Figure 3). Once there is enough phosphate for its growth, it is unwilling to oxidize more phosphite to phosphate (Figure 5).

Figure 5. Supernatant phosphate concentrationof each strain
grown in SMW (P, +3 valence), expressed as milligrams of phosphorus per liter.

For MRP, the situation is totally different. As shown in Figures 3 and 4, the biomass yield (expressed as maximum OD600) of MRP almost equals to that of MR, whereas the amount of phosphite that consumed by MRP was much more than that by MR. This portion of phosphorus is significantly beyond cellular growth requirements, resulting in a higher intracellular phosphorus content (Figure 6).

Figure 6. Intracellular phosphorus content of each strain grown in SMW (P, +3 valence),
expressed as milligrams of phosphorus per gram of dry weight (mg of P/g of DW).

2.2 Intracellular intermediate — polyphosphate (polyP)

We next confirmed that the extra portion of phosphate are stored in the form of the intermediate, polyP, in MRP cells. The presence of intracellular polyP was first examined by light microscopy. After staining, blue-purple to blue-black polyP granules in MRP cells can be easily visualized with an optical microscope (Figure 7).

Figure 7. Light microscopy images of stained cells that sampled from
SMW (P, +3 valence). PolyP granules appear blue-black, and polyP-free cells appear blue. Scale bar, 5 μm.

Based on the principle of our project, synthesis of polyP was catalyzed by PPK-M in the RPD+PPK-M-cassette using cellular ATP as the substrate [6]. Investigations performed on ATP metabolic pathway confirmed that, compared with MR, MRP actively redirected a substantial proportion of cellular ATP to polyP synthesis (Figure 8). Given that one phosphoric acid radical of ATP is deprived by PPK-M for polyP synthesis, regeneration of in vivo ATP necessitated the uptake of exogenous phosphorus [7, 8]. That is why MRP can oxidize exogenous phosphite to produce phosphate continuously (Figure 4).

Figure 8. Intracellular ATP levels of MR and MRP grown in SMW (P, +3 valence) as a function of OD600.

To validate the observed granules are indeed polyP, we extracted them (Figure 9) and then subjected them to gel assay (Figure 10).

Figure 9. Photographs of concentrated MRP cells and polyP extracted from MRP cells.

TBE-Urea PAGE analysis confirmed that they are mixtures of polyP molecules of different chain-length (Figure 10).

Figure 10. TBE-Urea PAGE analysis of polyP extracted from MRP at given time points.
Extracts of MR and MP served as the control. 50 bp DNA ladder and polyP molecules of given chain-length served
as the markers. Left panel, DAPI stained gel before photobleaching. Right panel, the same gel but imaged after photo-
-bleaching. PolyP appears as dark bands on the gel, because DAPI bound to polyP photobleached more quickly than that bound to DNA.

2.3 Extracellular final product — phosphate

Phosphate rather than polyP is the final product that we want to manufacture, therefore we have to recycle phosphate from MRP. Under anaerobic conditions, polyP can be hydrolyzed by the native exopolyphosphatase in E. coli and released back into medium in the form of phosphate [9, 10]. Based on this trait, we collected and concentrated 100 mL of MRP cells to 10 mL and then subjected them to anaerobic phosphate release. After release, we successfully obtained phosphate in the form of high-concentration solution (Figure 11).

Figure 11. Determination of supernatant phosphate that released by enriched MRP cells over time.
Phosphate + chromogenic reagent: blue; Phosphite + chromogenic reagent: no color development.

Part 3 Provide the heterotrophic chassis with oxygen and assimilable carbon source for phosphate manufacture on terrestrial planets

As noted above, our strategy involves the energy metabolism of the heterotrophic E. coli, for which the energy is essentially derived from the assimilation of glucose in SMW. However, all kinds of organic matters including glucose may be not available on terrestrial planets. Moreover, it is well known that polyP synthesis happens under aerobic conditions [11], which means that this process consumes oxygen. We solve these problems by the introduction of blue-green algae, Microcystis aeruginosa FACHB-469 (a M. aeruginosa strain incapable of producing cyanotoxins [12, 13]), hereafter abbreviated as algae.

3.1 Oxygen produced from photosynthesis by algae

Concurrent with carbon fixation is the release of oxygen, these oxygen bubbles can be easily visualized by the naked eye when the algae was subjected to stilling culture (Figure 12).

Figure 12. Oxygen produced from the photosynthesis of algae.

3.2 Algae lysate preparation

Due to the antagonistic action between bacteria and blue-green algae [14], blue-green algae cannot be used to feed E. coli directly. Therefore, a pretreatment must be performed to convert live algae to bioassimilable organic matters. For detailed information on how we develop the pretreatment method, please refer to the Proof of Concept. Two 250 mL Erlenmeyer flasks each containing 100 mL of algae culture were subjected to heat using electric heater (Figure 13). After 5 min of boiling, heating was stopped and the lysates were let to cool to room temperature.

Figure 13. Algae lysate prepared by heating.

To facilitate later test on E. coli growth, one bottle of lysate was clarified by centrifugation so as to gain a better display (Figure 14).

Figure 14. Comparison of algae lysate with or without cell debris.

3.3 Algae lysate utilization test

After the addition of phosphite, the algae lysate was subjected to MRP growth test. Compared with the biomass yield from SMW, MRP grew even better in the algae lysate (Figures 3 and 15). After culturing, the clarified lysate became turbid and this phenomenon can be easily visualized by the naked eye (Figure 15).

Figure 15. Comparison of the maximum biomass yield obtained from SMW (P, +3 valence) and clarified algae lysate.

For MRP that grown in the lysate without removing algae debris, it is not appropriate to monitor bacterial grown by optical density measurements. Therefore, we determined the colony forming units (CFU) to compare the difference on growth of MRP in these two kinds of lysate. The results showed that approximately 20% increase in biomass yield was further achieved when cultured using lysate with debris (Figure 16). This result implicated that, as far as MRP cultivation was concerned, it is not necessary to remove the debris when the algae lysate was adopted as the medium.

Figure 16. Comparison of the maximum biomass yield of MRP that grown in algae lysate with and without cell debris.

Next, to test whether phosphite was oxidized to phosphate by MRP that cultured using the algae lysate, we performed polyP staining. Microscopic observation showed that, as is the situation found with SMW cultivation, intermediate polyP accumulated in each MRP cell in the form of polyP granules (Figure 17). By this time, we confirmed that the algae lysate can be used to support not only the growth of MRP, but also the phosphate production pathway which it was engineered.

Figure 17. Light microscopy images of stained MRP cells that grow in algae lysate with debris. Red arrow, algae cell debris. Scale bar, 5 μm.

Part 4 Bench-scale phosphate production based on solar-energy driven sequence batch bioreactor

As a manufacture project, we devote ourselves to producing phosphate using phosphite as the substrate. Therefore, as the last part of our experiment, we initiated a bench-scale trial of real phosphate production. To achieve this trial in the context of terrestrial planets’ environment, we designed the suitable device, a solar-energy driven sequence batch bioreactor (Figure 18). Detailed information on design principle, configuration, and running regime, please refer to the Design and Hardware parts.

Figure 18. Photograph depicting the profile of solar-energy driven sequence batch bioreactor.

After three cycles, the phosphite from approximately 100L of algae lysate was almost fully taken up by MRP and converted to the intermediate, polyP. After a further membrane concentration, 9L of an enriched MRP cell was obtained and then subjected to anaerobic phosphate release test (Figure 19). At the end of the phosphate release phase, a cell suspension with supernatant phosphate concentrations of approximately 90 mg of P/L was formed.

Figure 19. Phosphate recovered from concentrated MRP cells, color
development test was performed on the solution of powdered solid for better display.

Part 5 Protocol

Molecular cloning and culture medium

All DNA fragments (either chemically synthesized or derived from the genomic DNA of E. coli K12) encoding the enzymes of interest were amplified using Q5 High-Fidelity DNA Polymerase. Ligation of PCR amplified fragments and the desired linearized vector was performed with In-Fusion HD Cloning Kit. After confirmation by sequencing, the plasmid was incorporated into E. coli K12 via electroporation. The positive colony was selected from LB agar plates supplemented with 50 mg/L kanamycin. Synthetic municipal wastewater (SMW) medium was prepared according to the method described by Bassin et al.[5] and contained per liter 300 mg of glucose, 100 mg of tryptone, 50 mg of sodium chloride, 110 mg of magnesium sulfate, 180 mg of ammonium chloride, and 1 mg of yeast extract. The phosphorus concentration in SMW was set at 20 mg of phosphorus per liter (mg-p/L) by the addition of 88 mg of potassium dihydrogen phosphate or 88 mg of sodium phosphite per liter.

qRT-PCR

Bacterial cells (equivalent to 10 mL culture with an OD600 value of 1) collected from SMW medium were pelleted by centrifugation and washed twice with 20 mM HEPES buffer (pH 7.5). Washed cells were homogenized in 1 ml TRIzol LS reagent by pipetting followed with 10 min of incubation at room temperature. Total RNA was then extracted per the manufacturer’s instructions. 1 mg qualified total RNA was subjected to reverse transcription with a PrimeScript RT reagent kit with gDNA Eraser. qRT-PCR of the resulting cDNA was performed on a CFX Connect Real-Time PCR Detection System with a SYBR Premix Ex Taq kit. Standard curves of cDNA dilutions were used to determine the PCR efficiency. An expression data analysis was performed by the Pfaffl method of relative quantification using CFX Manager software.

Supernatant phosphite and phosphate measurement

For phosphate concentration determination, 1 mL culture from SMW was clarified by centrifugation at 12,000 rpm for 1 min. Then, 200 μL clarified supernatant was subjected to phosphate quantification by ammonium molybdate spectrophotometry [15]. For phosphite concentration determination, 500 μL clarified supernatant was first digested by potassium persulfate, under which circumstances phosphite was transformed to phosphate. After quantification by ammonium molybdate spectrophotometry, phosphite concentration was calculated by subtracting pre-determined phosphate concentration.

Intracellular phosphorus content determination

E. coli K12 cells cultured using SMW were pelleted by centrifugation at 12,000 rpm for 5 min. Cell dry weight was measured after these pellets were dried at 100°C for 12 h. The dry weight was then correlated to OD600 by standard curve construction. For intracellular phosphorus assay, equal amount of engineered E. coli K12 cells (determined by OD600 and culture volume) sampled from SMW medium was digested by potassium persulfate, under which circumstances all the phosphorous compounds were transformed to phosphate. After that, phosphate was quantified by ammonium molybdate spectrophotometry. Then, intracellular phosphorus content was calculated using the determined cell dry weight and phosphate.

Microscopic observation

The presence of intracellular polyP granules was examined by quick polyP staining method adapted from that of Albert [16]. PolyP staining was performed by mixing bacterial culture and toluidine blue solution (2% w/v) of equal volume. After 5 min of incubation at room temperature, smear the mixture on the preheated glass slide. The excess dye was then washed by water. Dried slide was subjected to light microscopy observation.

TBE-Urea PAGE analysis

Intracellular polyP formation of engineered E. coli K12 was verified by TBE-urea PAGE analysis as described by Ursula et al [17]. Briefly, bacterial cells collected from 1 mL SMW medium were resuspended in 50 µL 20 mM HEPES (pH 7.5) and boiled at 100°C for 10 min. After centrifugation at 12,000 rpm for 10 min, the supernatant was mixed 1:1 with 2 × TBE-urea sample buffer. Samples were then loaded on a 15% TBE-urea PAGE gel that was manually casted with 40% acrylamide/bis-acrylamide (29:1) and urea. PolyP of given chain-length (kindly provided by Dr. Toshikazu Shiba, Regenetiss, Japan) served as the marker. Subsequently, electrophoresis was carried out as described by Heike et al. [18] and “negative” DAPI staining was performed according to Morrissey et al. [19] on the UV sample tray of ChemiDoc Touch Imaging System. Because DAPI bound to polyP photobleached more quickly than that bound to DNA, polyP appears as dark bands on the gel.

Cellular ATP assay

In vivo ATP measurements were performed with a modified protocol described by Gray et al [17]. Briefly, at the indicated OD600, bacterial cells that collected from 100 μL SMW were washed twice by 20 mM HEPES (pH 7.5). After that, washed cells were resuspended in 100 μL of the same buffer and incubated at 99°C for 5 min. The samples were cooled on ice and the total cellular ATP was assayed with the luminescent ATP detection assay kit.

Extraction of polyP

Engineered E. coli K12 cells with intracellular polyP granules were collected by centrifugation at 12,000 rpm for 5 min. The resulted cell pellet was washed and resuspended in pure water. After ultrasonic decomposition, homogenous cell lysate was incubated at 99°C for 1 h to release protein bounded polyP molecules. Then, supernatant that is rich in polyP was pooled by centrifugation at 12,000 rpm for 10 min. PolyP was recovered after lyophilization.

Algae cultivation and algae lysate preparation

The non-toxic blue-green algae, Microcystis aeruginosa FACHB-469 were purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). BG11 medium was adopted for the routine maintenance of this algae [20]. After cultivation, algae cells were harvested by centrifugation at 2,000 rpm for 5 min. The harvested algae cells (i.e., concentrated algae cells) can be stored at -20°C for long term preservation, and repeated freeze-thaw cycles have no detectable effect on the usage of it as a raw material for natural medium preparation, except that the color will get darker. To prepare algae lysate, 5 g concentrated algae cells were resuspended in 100 mL water, after which the suspension was boiled for 5 min. Phosphate or phosphite was then added to give a final phosphorus concentration of 20 mg/L before it was used as the culture medium. For algae cultivation in Hardware, phosphate that we manufactured and carbon dioxide served as the phosphorus source and carbon source, respectively, and the corresponding components were omitted from BG11 medium.

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