Part:BBa_K5108009

creA - crnA operon for creatinine metabolization

P. fluorescens creatinine amidohydrolase and creatinase ORFs with RBS

Contents
1. Usage and Biology
2. Sequence and Features
3. Characterization and Measurements
   1. SDS-PAGE
   2. Growth analysis
   3. Consumption analysis of creatinine by NMR spectroscopy
4. Conclusion and Perspectives
5. References

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1329
  Illegal NgoMIV site found at 1931
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 249
  Illegal BsaI site found at 1036
  Illegal BsaI site found at 1467
  Illegal BsaI.rc site found at 1815
  

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).

Figure 1: Metabolic pathway of the creatinine degradation in Pseudomonas putida.

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.

Figure 2: Schematic of the cloning strategy for pSEVA438-Ptet-creA-crnA plasmid.

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 3: Restriction digest of pSEVA438-Ptet-creA-crnA plasmid. The plasmid was digested with EcoRI and HindIII separately or in combination. The expected (left) and experimental (right) digestion patterns are shown.

Characterization and Measurements

SDS-PAGE

To assess protein production, the strain was grown on M9 minimal medium with glucose (28 mM), with or without 0.5 mM of m-toluic acid (as inducer of the Pm promoter). P. fluorescens transformed with a pSEVA438-MBPeGFP plasmid was used as positive control of Pm promoter's inducibility in P. fluorescens. This 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 4. 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 produced at higher yield 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 was 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.

Figure 4: SDS-PAGE of soluble and insoluble protein fractions from cultures of Pseudomonas fluorescens transformed with pSEVA438-MBPeGFP or pSEVA438-Ptet-creA-crnA. P. fluorescens was cultured with and without inducer. Arrows show expected size of MBPeGFP, creatinine amidohydrolase (CrnA) and creatinase (CreA).

Growth analysis

WT P. fluorescens SBW25 was transformed with either the empty plasmid pSEVA438-Ptet as a control or the plasmid pSEVA438-Ptet-creA-crnA, or no plasmid as a second control. The three strains were cultured in M9 minimal medium without NH₄Cl (as it would provide a nitrogen source) and supplemented either with creatinine (44 mM), creatine (38 mM), or sarcosine (67 mM). The inducer m-toluic acid was added at 0.5 mM. The growth curves for each condition are presented in Figure 5.

Figure 5: Growth of the engineered Pseudomonas fluorescens SBW25 strain with creA-crnA operon on M9 minimal medium complemented with creatinine, creatine or sarcosine. Optical density (OD) at 600 nm was measured every 30 minutes. The mean value of the biological and technical replicates for each condition is shown (n ≥ 3).

No growth was observed in the WT and the negative control strains when cultured with creatinine, confirming that our strain P. fluorescens SBW25 does not naturally metabolize this compound. In contrast, 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 (Figure 5). These physiology results indicate that the crnA gene is well expressed, although the yield of protein production was too low to be detected by SDS-PAGE (Figure 4).

Consumption analysis of creatinine by NMR spectroscopy

Supernatants from bacterial cultures were collected during the exponential growth phase 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 6). Starting at an initial concentration of 26 mM, the creatinine uptake rate was established at 3.58 mmol.h⁻¹.gDW⁻¹. These results demonstrate that P. fluorescens carrying the creA-crnA plasmid can degrade creatinine and utilize it as a sole source of carbon and nitrogen.

Figure 6: Uptake of creatinine by the transformed pSEVA438-Ptet-creA-crnA Pseudomonas fluorescens SBW25 strain on M9 minimal medium complemented with creatinine. Samples of supernatants were taken every hour during the exponential phase and creatine concentration was analyzed by 500 MHz NMR spectroscopy. Simulation of creatine uptake (dashed line) was done with Physiofit. The shaded area indicates the 95% confidence interval.

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

Conclusion and Perspectives

In conclusion, our creA-crnA operon enables the metabolization of a urine compound by the plant growth-promoting rhizobacteria, Pseudomonas fluorescens. The cloning of these two genes into plasmid pSEVA438 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) suggests that further optimization may be needed to improve the pathway efficiency. For instance, we suggest to increase the activity of creatinase (CreA)(iGEM ToulouseINSA-UPS 2024).

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