Difference between revisions of "Part:BBa K5108009"

Revision as of 16:53, 26 September 2024

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 with a pSEVA438-MBPeGFP plasmid was used as positive control of Pm promoter's inducibility in P. fluorescens. 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.

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

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.