Difference between revisions of "Part:BBa K5108009"

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creA - crnA operon for creatinine metabolization

P. fluorescens creatinine amidohydrolase and creatinase ORFs with RBS

Usage and Biology

Creatinine is a urinary human waste, rich in carbon and nitrogen. 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 (Figure 1).

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.

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. 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).

Characterization and Measurements

SDS-PAGE

To verify that there was protein production the strain was grown on M9 minimal medium with glucose (28 mM), with and without 0.5 mM of m-toluic acid (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 SBW25. This last construct encodes the Maltose-Binding Protein (MBP) fused to enhanced Green Fluorescent Protein (eGFP) under the control of the Pm promoter.

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 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.

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 5.

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.

NMR results

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 6, 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 (5 g/L). 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 5.
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.

Creatine degradation:

Molecular Modeling

Conclusion and Perspectives

References

1. Xiao, D., Zhang, W., Guo, W., Liu, Y., Hu, C., Guo, S., Kang, Z., Xu, X., Ma, C., Gao, C., & Xu, P. 2021. A D-2-hydroxyglutarate biosensor based on specific transcriptional regulator DhdR. Nature Communications 12, 7108.
2. Jezek, P. 2020. 2-Hydroxyglutarate in Cancer Cells. Antioxid Redox Signal, 33(13),903-926.