Part:BBa_K5108009

RBS aprA CHA0 - creA - RBS hcnA CHA0 - crnA

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 sarcosine, creatine and creatinine by NMR spectroscopy
4. Modeling
5. Conclusion and Perspectives
6. 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 carbon compound rich in nitrogen excreted in human urine [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 carbon and nitrogen source to support the growth of Pseudomonas fluorescens, which serves as biostimulant for plant. Certain species of Pseudomonas, such as Pseudomonas putida can degrade creatinine and use it as carbon and nitrogen sources to ensure its growth. There is no bibliography on this pathway being present 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 in the same operon. The first catalyzes the hydrolysis of creatinine into creatine. Then, the creatinase catalyzes the hydrolysis of creatine into sarcosine and urea. Finally, in P. putida, sarcosine is degraded by a sarcosine oxidase to join the glycine metabolism [2] (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 ensure the 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 from P. putida, and two RBS (BBa_K5108006, BBa_K5108007) allowing their expression in P. fluorescens. This part was expressed under the Pm promoter control into the pSEVA438-Ptet vector (Figure 2). The constitutive repression by the XylS is lifted by the m-toluic acid. We demonstrated efficient enzyme activity to support P. fluorescens growth.

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

To create the functional vector containing the creA-crnA operon, the cloning of the creA-crnA synthetized gBlocks into the pSEVA438-Ptet linearized vector was performed following In-Fusion Assembly (Takara). Figure 3 demonstrates the successful cloning by restriction digest with EcoRI and HindIII enzymes (New England Biolabs R3101S, R3104S) (Figure 3). The construct was confirmed by Sanger sequencing (Genewyz, Figure 4).

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.

Figure 4: creA-crnA locus’ sequencing of pSEVA438-Ptet-creA-crnA plasmid. The creA-crnA operon was sequenced by two Sanger sequencing using two flanking primers.

Characterization and Measurements

SDS-PAGE

The pSEVA438-MBPeGFP plasmid, originally used in P. putida KT2440, was employed as positive control of Pm promoter's inducibility in P. fluorescens SBW25. This construct encodes the fusion protein MBPeGFP (Maltose-Binding Protein enhanced Green Fluorescent Protein) under the control of the Pm promoter. Based on the results of Vogeleer P. et al. (2024) [3], the pSEVA438-MBPeGFP- and pSEVA438-Ptet-creA-crnA-transformed P. fluorescens SBW25 strains were cultured in M9 minimal medium supplemented with glucose (28 mM), with or without 0.5 mM of m-toluic acid inducer. After incubation, a whole-protein extraction was performed for each strain to assess the level of expression, as well as the solubility of our proteins.

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 overproduced 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.

Figure 5: 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, m-toluic acid. Arrows show expected size of MBPeGFP, creatinine amidohydrolase (CrnA) and creatinase (CreA).

Growth analysis

To challenge the efficiency of our construction, P. fluorescens SBW25 WT 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.

Figure 6: Growth of the engineered Pseudomonas fluorescens SBW25 strain with creA-crnA operon on M9 minimal medium complemented with creatinine, creatine or sarcosine. Measurement of the OD at 600 nm every 30 minutes. The mean value of the biological and technical replicates for each condition is shown (n ≥ 3). The term plasmid refers to pSEVA438-Ptet.

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. 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.

In analogy with the results obtained with creatinine, neither the WT nor the negative control strains grew on creatine. However, the engineered strain with creA-crnA operon showed moderate growth, confirming that the transformed bacteria could also metabolize creatine. The same growth has been observed for growth on creatine as a sole source of carbon and nitrogen.

However, the growth rate on sarcosine (0.10 h⁻¹) was higher than on creatinine, indicating that the pathway for creatinine degradation may not be as efficient as for sarcosine. This suggests a need for optimization of the metabolic pathway, as differences in growth rates point to potential bottlenecks or suboptimal expression of the enzymes involved in creatinine degradation.

Using sarcosine as a substrate, all three strains could grow. Growth rate of the strains transformed with the empty plasmid (0.11 h⁻¹) or with the pSEVA438-Ptet-creA-crnA plasmid (0.10 h⁻¹) was lower than that of the WT strain (0.15 h⁻¹), indicating that plasmid introduction had an adverse effect on the growth rate. This was not surprising, as the production of CreA and CrnA may impose a metabolic burden on the bacteria, along with the cost of maintaining antibiotic resistance.

Consumption analysis of sarcosine, creatine and 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.

Sarcosine degradation:

As shown in Figure 7, the experimental data reveals a clear decrease in sarcosine concentration over time. The initial concentration of sarcosine started at approximately 40 mM, although the expected concentration based on initial conditions was 56 mM. NMR analysis was conducted on the supernatants collected during the exponential growth phase of the bacteria. A sarcosine uptake rate of 3.6 mmol.h⁻¹.gDW⁻¹ was obtained, while the measured growth rate under this condition was 0.105 h⁻¹. The lower growth rate observed in the transformed P. fluorescens strain on sarcosine (0.105 h⁻¹) compared to the WT strain (0.186 h⁻¹) in the same condition (growth in Erlenmeyer flasks) can likely be attributed to the metabolic burden imposed by the plasmid carrying the creA-crnA operon. This effect was similarly observed in microplate experiments, where the growth rate of the transformed strain (0.10 h⁻¹) was also lower than that of the WT strain (0.15 h⁻¹) as presented in Figure 6.
To validate the predictability of our metabolic model, we used the experimentally measured sarcosine uptake rate as an input parameter and the model predicted a growth rate of 0.113 h⁻¹, which is in perfect agreement with the experimentally observed rate of 0.105 h⁻¹.

Figure 7: Uptake of sarcosine by the transformed pSEVA438-Ptet-creA-crnA Pseudomonas fluorescens SBW25 strain on M9 minimal medium complemented with sarcosine. Samples of supernatants were taken every hour during the exponential phase and sarcosine concentration was analyzed by 500 MHz NMR spectrometry. Simulation of sarcosine uptake (dashed line) was done with Physiofit with the following characteristics. The shaded area indicates the 95% confidence interval. The growth rate on sarcosine is 0.105 h-1 (n=1).

Creatine degradation:

The degradation of creatine was also monitored through NMR (Figure 8). The initial creatine concentration was lower than expected (18 mM, with an expected value of 38 mM), but similar trends were observed in terms of degradation. The experimental data fit well with the simulated predictions, further validating our metabolic model for creatine degradation. A creatine uptake rate of 2.05 mmol.h⁻¹.gDW⁻¹ was obtained, while the measured growth rate under this condition was 0.054 h⁻¹. To validate the predictability of our metabolic model, we also used the experimentally measured sarcosine uptake rate as an input parameter and the model predicted a growth rate of 0.078 h⁻¹, which remains in agreement with the experimentally observed rate of 0.054 h⁻¹.

Figure 8: Uptake of creatine by the transformed pSEVA438-Ptet-creA-crnA Pseudomonas fluorescens SBW25 strain on M9 minimal medium complemented with creatine. Samples of supernatants were taken every hour during the exponential phase and creatine concentration was analyzed by 500 MHz NMR spectrometry. Simulation of creatine uptake (dashed line) was done with Physiofit with the following characteristics. The shaded area indicates the 95% confidence interval. The growth rate on creatine is 0.054 h⁻¹ (n=1).

Creatinine degradation:

Lastly, the degradation of creatinine was analyzed (Figure 9). Starting at an initial concentration of 26 mM (with an expected value of 44 mM), creatinine degradation followed a similar trajectory to the other compounds. However, the degradation rate of creatinine was slower than that of sarcosine and creatine suggesting a lack of expression of the gene crnA as discussed in the precedent part. A creatine uptake rate of 3.58 mmol.h⁻¹.gDW⁻¹ was obtained, while the measured growth rate under this condition was 0.034 h⁻¹. To validate the predictability of our metabolic model, we also used the experimentally measured sarcosine uptake rate as an input parameter and the model predicted a growth rate of 0.014 h⁻¹, which is this time quite high in comparison with the experimentally observed rate of 0.034 h⁻¹.

Figure 9: 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 spectrometry. Simulation of creatine uptake (dashed line) was done with Physiofit with the following characteristics. The shaded area indicates the 95% confidence interval. The growth rate on creatinine is 0.034 h⁻¹ (n=1).

For the three experiments, we observed lower-than-expected initial substrate concentrations. It could be due to incomplete errors during preparation or measurement. While this raises questions about the accuracy of the initial concentrations, it does not undermine the validity of the results as long as the degradation rates and growth patterns are consistent across experiments and closely match model predictions. Further replicates or adjustments to preparation protocols could have helped us clarify this issue.

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 to support its growth. 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.

Modeling

To further substantiate our efforts in engineering this pathway we put into place an effective modeling strategy. Through the reconstruction in silico of a P. fluorescens Genomic Scale Metabolic Model (GSMM), we were able to perform Flux Balance Analysis (FBA) and therefore accurately predict and characterize metabolic behavior.

The obtained resultats are presented in Figure 5. The growth rate on sarcosine, creatine, and creatinine is significantly higher than growth rate on glycine for the same uptake rate, this suggest an effect on growth caused by some of the byproducts of the pathway (Figure 5A). Formaldehyde was constrained to be excreted at the rate with which it was formed (i.e. flux of sarcosine oxidation to glycine) (Figure 5B). When formaldehyde is not metabolized, growth rate is significantly more similar across the four substrates compared to when it is (Figure 5A).

Figure 5: In silico growth simulation on creatinine, creatine and sarcosine of Pseudomonas fluorescens. The uptake of substrates depending on the growth rate are represented with (A) and without (B) metabolization of formaldehyde formed by the pathway.

Specifically, by implementing the creatinine degradation pathway in silico, we validated our cloning design by revealing the presence of a sarcosine oxidase analogue into the WT strain. The prediction was tested in the lab and confirmed in silico results that P. fluorescens can grow in sarcosine. Furthermore, laboratory results provided growth rates and uptake rates on each substrate of the creatinine metabolization pathway. By constraining our model to the measured uptake rates, we compared the ideal growth rates predicted by the model to those measured in the lab. By doing so, we were able to confirm that growth rate on sarcosine and creatine is optimal, meaning that growth rates in vivo are close to ideal. Notably, optimality of growth on sarcosine is preserved between the WT and transformed strains, indicating that our engineering did not decrease optimality. These results were extremely valuable as they allowed us to use the model to understand data produced in the lab and make predictions about the dynamics of our synthetic pathway. Moreover, we predicted the native growth on sarcosine for which no information was available in literature.

Conclusion and Perspectives

Further studies will be needed to optimize the metabolic pathway for creatinine and to investigate the potential causes of lower degradation efficiency.

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