Difference between revisions of "Part:BBa K5108009"

Revision as of 14:24, 26 September 2024

creA - crnA operon for creatinine metabolization

P. fluorescens creatinine amidohydrolase and creatinase ORFs with RBS

Sequence and Features

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).

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

Sequence and Features (Successful Assembly of pSEVA438-Ptet-creA-crnA plasmid)

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: Schematic 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. 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.