Pre-experiment results

1. Changes in Inorganic Phosphate During Electricity Generation

We culture Shewanella in standard Luria-Bertani medium and measure the inorganic phosphate (Pi) concentration in the medium at 0h, 12h, 24h, and 36h. The results show a decrease in phosphorus concentration in the medium during the electricity generation process (Fig. 1), suggesting that the bacteria require Pi during electrocatalysis.

2. Effect of Pi on Electricity Generation

Next, to investigate the effect of Pi concentration on electron transfer and electricity generation, we culture Shewanella in Pi-full and Pi-free medium. Throughout the culture process, we continuously monitor the output current. It turns out that the output current in the Pi-free environment is only 40.8 ± 4.74 µA/cm², which is 60.65% lower than the output current in the Pi-full environment (103.69 ± 8.88 µA/cm²) (Fig. 2), indicating that Pi is crucial for electron transfer.

1. Cycle1: Inducing enzymes polyphosphate kinase 1 (PPK1)

1.1 Pi Concentration and Electricity Generation Capability Testing

Due to pre-experiment findings indicating that Shewanella requires Pi for electron transfer, we introduce PPK1 enzyme into Shewanella. The main function of PPK1 is to polymerize Pi into polyP, thereby promoting polyphosphate accumulation. We connect this gene to the ribosome binding site(RBS) [Registry code: BBa-B0034] to enhance PPK1 gene expression. We refer to this engineered strain as SPK1.

Detection results show that SPK1’s polyphosphate production capability is four times greater than that of the wild-type strain(WT), reaching 139.6 mg-P/L. However, the current density is only 23% of that of the WT, measuring just 23.6 µA cm² (Fig. 3). This level of electricity production efficiency is unsatisfactory, prompting us to analyze the reasons for SPK1's weak electricity generation capacity.

1.2 Analysis of the Factors Contributing to the Reduced Electricity Generation Capability of SPK1

To investigate the factors of SPK1's reduced electricity generation capability, we examine SPK1 strain using Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM). SEM images show that SPK1 bacteria exhibit a less smooth morphology compared to WT (Fig. 4).

TEM results indicate that SPK1 bacteria appear to be "cut in half" (Fig. 5). A possible reason is that the high expression of PPK1 may lead to an accumulation of excessive negative charges within the bacterial cells.

In summary, morphological characterization of the bacteria shows that strain with high Pi concentration exhibit abnormal morphology, suggesting that pre-conditioning bacteria to a high polyphosphate state is detrimental to rapid electron transfer.

2. Cycle2: Engineered SPPK1 Strains with Different RBS Designs

2.1 Plasmid Construction and Transformation

Due to the high polyphosphate state being unfavorable for rapid electron transfer, we explore whether adjusting the strength of the RBS to modify PPK1 expression can restore electricity production capacity.

We construct three kinds of plasmids with varying strengths of RBS linked to the gene encoding PPK1 and transform them into wild-type Shewanella. The RBS serial numbers used are BBa-B0031, BBa-B0032, and BBa-B0034, with BBa-B0031 having the lowest strength and BBa-B0034 having the highest strength. The resulting strains are named SPK3, SPK2, and SPK1, respectively (Fig. 6).

We then perform a series of experiments to verify the successful construction and expression of the plasmids in Shewanella. DNA agarose gel electrophoresis results show that we obtain three kinds of plasmids with different RBS sequences, each approximately 2.1 kb in size (Fig. 7).

Next, we transform the plasmids into wild-type Shewanella, express it, and perform colony PCR and protein extraction for SDS-PAGE. The results show that PPK1 is successfully introduced into Shewanella for replication (Fig. 8).

As the strength of the RBS increases, the expression level of the PPK1 protein (approximately 70 kD) also increases (Fig. 9).

2.2 Pi Concentration and Electricity Generation Capability Testing

As we expected, as the strength of the RBS decreases, the electrochemical activity of Shewanella significantly increases, while polyphosphate capacity decreases. The electricity production capacity of SPK3 is approximately 351.5% higher than that of SPK1. However, the polyphosphate capacity of SPK3 is only about 54.0% lower than that of SPK1, which is not as critical when considering the issue of weaker electricity generation(Fig. 10). Nonetheless, we continue to explore better synthetic biology approaches to further optimize both electricity generation and polyphosphate accumulation capabilities.

2.3 The Growth Status of the Engineered Strains

To investigate the growth status of SPK1, SPK2, and SPK3, we culture them in LB medium for 15 hours, measuring the OD600 every three hours. A higher OD600 value indicates a greater number of bacteria. The results show that the bacteria numbers of SPK3, SPK2, and SPK1 are significantly lower than that of the WT strain, with SPK1 exhibiting the least number, indicating that they are in an unfavorable growth state(Fig. 11). This aligns with our previous SEM and TEM results (Fig. 4-5), collectively suggesting that preconditioning bacteria to a high polyphosphate state is detrimental to their growth.

3. Cycle3: Engineered Strains with Different Hydrolytic Enzyme

3.1 Plasmid Construction and Transformation

We hypothesize that regulating the polymerization of phosphates in bacteria may enable control over their electron transfer efficiency. Since PolyP mainly participates in the polymerization and hydrolysis of inorganic phosphate, along with predictions from dry experimental models, our subsequent synthetic biology strategy is to construct engineered strains based on five polyphosphate-metabolizing enzymes(PPX, PPK2, NADK, PAP, PPN1). The five constructed strains are named SPPX, SPPK2, SNADK, SPAP, and SPPN1.

Similarly, we conduct a series of experiments to verify the successful construction of our plasmids and their introduction into Shewanella. The results of DNA agarose gel electrophoresis show that we obtain gene fragments for each hydrolase, ranging in size from 1.0 to 2.0 kb. The bands at the bottom of the PPN1 and PAP lanes are primer dimers, which do not interfere with our experiments(Fig. 12).

Next, we transform the plasmids into wild-type Shewanella, express them, and perform colony PCR. The results also indicate that the genes of each enzyme are successfully introduced into Shewanella for replication (Fig. 13).

3.2 Pi Concentration and Electricity Generation Capability Testing

After culturing in standard LB medium for 12 hours, we measure the remaining Pi concentration in the medium to indirectly determine the polyphosphate levels of each group. The results indicate that the SPPK2 and SNADK strains have higher polyphosphate accumulation efficiency. Meanwhile, half-cell experiments show increased electron transfer activity, with the SPPK2 strain exhibiting a current density of 137.4 ± 16.34 µA/cm² and the SNADK strain showing 134.56 ± 17.01 µA/cm² (Fig. 14).

3.3 Detection of Cellular Metabolic Activity

The intracellular ATP content and NADH/NAD+ level in the SPPK2 and SNADK strains are higher than WT strain(Fig. 15), indicating active intracellular metabolism. Therefore, we believe that the increase in electron transfer activity is due to the elevated intracellular metabolic levels of the engineered strains.

4. Cycle4: Combination of PPK2 and NADK enzymes

4.1 Performance of SPPK2-NADK strain

Since both PPK2 and NADK exhibit good performance individually, we attempt to combine these two hydrolases in series to see if there will be improved results. Since the PPK2 gene is approximately 1.1 kb and the NADK gene is about 1.0 kb, the PPK2-NADK construct should be approximately 2.1 kb. The colony PCR results show a band at about 2.1 kb, confirming that we successfully introduce the plasmid containing PPK2-NADK into Shewanella (Fig. 16).

Half-cell experiments show increased electron transfer activity, with the current density of the SPPK2-NADK strain measuring 164.2 ± 17.64 µA/cm², approximately 30 µA/cm² higher than that of the SPPK2 and SNADK strains (Fig. 17).

With the introduction of PPK2-NADK, the phosphorus accumulation capacity of the SPPK2-NADK strain was also significantly improved.(Fig. 18)

Similarly, the intracellular ATP content and NADH/NAD⁺ level of the SPPK2-NADK strain are significantly higher, reaching 0.28 µmol/10⁸ cells and 1.79, which are 3.59 times and 5.42 times that of the WT strain (Fig. 19).

4.2 Pi Removal Efficiency Verification

The experimental data above indicate that the SPPK2-NADK engineered strain has a significant advantage in electricity production compared to other strains. Given that our goal is to construct an engineered strain that can efficiently catalyze electricity generation while also achieving polyphosphate accumulation,we used M9 cultural medium, which has a higher phosphorus content, in order to explore its application prospects. We then examine the Pi levels at 12, 24, and 36 hours. The results show that the phosphate removal rate increases by 36.8–48.5% during the electron transfer process (Fig. 20).

This demonstrates that the engineered strain SPPK2-NADK successfully facilitates both phosphate removal and electrocatalysis simultaneously.

4.3 Other Electrochemical Parameters Measurement

To explore the specific electrochemical performance of the SPPK2-NADK strain, we conduct the following experiments:

Firstly, we use cyclic voltammetry (CV) and linear sweep voltammetry (LSV) techniques for testing. The CV curve shows higher redox activity in the SPPK2-NADK strain (Fig. 21);

the LSV curve indicates lower internal resistance in the MFC cells of the SPPK2-NADK strain (Fig. 22).

Next, we measure the relative output power. The power density results show that the SPPK2-NADK strain has a maximum output power of 243.77 ± 25.2 mW/m², which is 2.32 times higher than the WT strain’s output power density (105.06 ± 11.72 mW/m²) (Fig. 23).

These results indicate that the electrochemical performance of the SPPK2-NADK strain is significantly better than that of the WT strain.

5. Supplementary Figures

We constructed a plasmid using a double enzyme digestion method, where the cleavage site is EcoR I and Not I. It is about 5000 bp.

6. Reference

[1]: Zhang H, Ishige K, Kornberg A. A polyphosphate kinase (PPK2) widely conserved in bacteria. Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16678-83. doi: 10.1073/pnas.262655199. Epub 2002 Dec 16. PMID: 12486232; PMCID: PMC139203.

[2]: Mori S, Yamasaki M, Maruyama Y, Momma K, Kawai S, Hashimoto W, Mikami B, Murata K. Crystallographic studies of Mycobacterium tuberculosis polyphosphate/ATP-NAD kinase complexed with NAD. J Biosci Bioeng. 2004;98(5):391-3. doi: 10.1016/S1389-1723(04)00302-0. PMID: 16233726.