Difference between revisions of "Part:BBa K5108009"

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RBS aprA CHA0 - creA - RBS hcnA CHA0 - crnA

P. fluorescens creatinine amidohydrolase and creatinase ORFs with RBS

Usage and Biology

Creatinine is a urinary human waste, rich in carbon and nitrogen [1]. During recent years countless research has been done on the topic of waste-upcycling and revalorization. Creatinine is one of the few human waste products still to be valorized during space missions. In our project, we wanted to use it as a carbon and nitrogen source to support the growth of Pseudomonas fluorescens, which serves as a biostimulant for plants. Certain species of Pseudomonas like Pseudomonas putida can degrade creatinine and use it as carbon and nitrogen sources to sustain growth. There is no bibliography on the presence of this pathway in P. fluorescens. The two enzymes permitting creatinine metabolization are creatinine amidohydrolase (CrnA, EC 3.5.2.10) and creatinase (CreA, EC 3.5.3.3), both expressed from an operon. CrnA catalyzes the hydrolysis of creatinine into creatine. Then, CreA catalyzes the hydrolysis of creatine into sarcosine and urea. Finally, in P. putida - but this was not reported in P. fluorescens - sarcosine is degraded by a sarcosine oxidase to enter the glycine metabolism (figure 1).

Sequence and Features

The part BBa_K5108009 permits the utilization of creatinine as sole carbon and nitrogen sources to sustain growth of P. fluorescens. This part is composed of the creatinase and creatinine amidohydrolase ORFs (creA BBa_K5108003, crnA BBa_K5108004) keeping the natural order found in P. putida in the form of an operon, and two RBS (BBa_K5108006, BBa_K5108007) enabling their expression in P. fluorescens. This part was expressed under control of the Pm promoter into the pSEVA438-Ptet vector (Figure 2). Repression by the XylS transcription factor can be lifted by addition of m-toluic acid.

To create the expression plasmid containing the creA-crnA operon, we cloned of the PCR-amplified creA-crnA gBlocks into the linearized pSEVA438-Ptet vector by In-Fusion Assembly (Takara, France). Figure 3 shows the expected digestion pattern using EcoRI and HindIII enzymes (New England Biolabs, France R3101S, R3104S). The construct was confirmed by Sanger sequencing (Genewyz, Germany, Figure 4).

Characterization and Measurements

SDS-PAGE

To assess protein production, the strain was grown on M9 minimal medium with glucose (28 mM), with and without 0.5 mM of m-toluic acid (as the Pm promoters inducer). A transformed P. fluorescens SBW25 with a pSEVA438-MBPeGFP plasmid was used as positive control of Pm promoter's inducibility in P. fluorescens. This last construct encodes a maltose-binding protein (MBP) fused to enhanced Green Fluorescent Protein (eGFP) under the control of the Pm promoter (gift from P. Vogeleer, [3]).

The obtained SDS-PAGE is presented in Figure 5. Both soluble and insoluble fractions contain MBPeGFP, with the majority of protein being in the soluble fraction independently of the presence of the inducer. Although transcriptional leakage was clearly observed without the inducer, MBPeGFP was more produced when the Pm promoter was activated with 0.5 mM of m-toluic acid, confirming the functionality of the Pm promoter in P. fluorescens. The presence of insoluble MBPeGFP can be caused by its overexpression leading to protein aggregation. SDS-PAGE analysis of the cell lysate derived from P. fluorescens transformed with pSEVA438-Ptet-creA-crnA revealed a clearly visible band at the expected size of CreA in both soluble and insoluble fractions when its expression is induced. As expected based on the leaky expression of MBPeGFP, CreA is also produced without the inducer in the soluble protein fraction. In contrast, there is no visible band at the expected size of CrnA, suggesting that the crnA gene is not or poorly expressed.

Growth analysis

To challenge the efficiency of our construction, WT P. fluorescens SBW25 was transformed with the empty plasmid pSEVA438-Ptet as a control or plasmid pSEVA438-Ptet-creA-crnA, or no plasmid as a second control. The three strains were all cultured in M9 minimal medium without NH4Cl and supplemented either with creatinine (44 mM), creatine (38 mM), or sarcosine (67 mM). The Pm inducer m-toluic acid was added at 0.5 mM into all the culture media. The growth curves of each condition are presented in Figure 6.

No growth was observed in either the WT or the negative control strains when cultured with creatinine, confirming that the WT P. fluorescens SBW25 strain does not naturally metabolize this compound. Using sarcosine as a substrate, all three strains could grow, as expected. Only the engineered strain harboring the creA and crnA genes exhibited clear growth on creatinine, demonstrating that the introduced metabolic pathway is functional with a growth rate of 0.06 h⁻¹. The transformed bacteria could also metabolize creatine.

Consumption analysis of creatinine by NMR spectroscopy

The engineered strain with creA-crnA operon was cultivated of in M9 minimal medium supplemented with creatinine (44 mM), creatine (38 mM), or sarcosine (67 mM). Supernatants during the exponential growth phase were collected and analyzed using 500 MHz NMR spectroscopy to monitor the concentrations of sarcosine, creatine, and creatinine over time. Growth rates and uptake rates were calculated using Physiofit.

The consumption of creatinine was analyzed (Figure 7). Starting at an initial concentration of 26 mM, the creatine uptake rate was established at 3.58 mmol.h⁻¹.gDW⁻¹.

These results demonstrate that P. fluorescens carrying the pSEVA438-Ptet-creA-crnA plasmid can degrade sarcosine, creatine, and creatinine, and utilize it as a sole source of carbon and nitrogen. The experimental data from NMR closely matched the simulations for sarcosine and creatine, confirming the functionality of our metabolic module for these compounds. Although creatinine degradation appeared less efficient, it was still observed, further supporting the module. Further studies will be needed to optimize the metabolic pathway for creatinine and to investigate the potential causes of lower degradation efficiency.

Similar results are obtained for creatine and sarcosine consumption (iGEM ToulouseINSA-UPS 2024).

Conclusion and Perspectives

In conclusion, our operon creA-crnA allows the metabolization of a urine compound by the plant growth-promoting rhizobacteria, P. fluorescens. The cloning of these two genes into plasmid pSEVA438-Ptet and transformation into our chassis bacteria allowed it to use creatinine as sole carbon and nitrogen source. However, the slower growth rates on creatinine (0.06 h⁻¹) compared to sarcosine (0.10 h⁻¹) suggest that further optimization may be needed to improve the pathway efficiency. For instance, we suggest to increase the activity of creatinase (CreA).

References

1. Washington IM & Van Hoosier G (2012) Chapter 3 - Clinical Biochemistry and Hematology. In The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents, Suckow MA Stevens KA & Wilson RP (eds) pp 57–116. Boston: Academic Press
2. Tsuru D, Oka I & Yoshimoto T (1976) Creatinine Decomposing Enzymes in Pseudomonas putida. Agricultural and Biological Chemistry 40: 1011–1018
3. Vogeleer P, Millard P, Arbulú A-SO, Pflüger-Grau K, Kremling A & Létisse F (2024) Metabolic impact of heterologous protein production in Pseudomonas putida: Insights into carbon and energy flux control. Metabolic Engineering 81: 26–37