Recombinant Pseudomonas putida Inorganic pyrophosphatase (ppa)

PPA in Pseudomonas putida

Pseudomonas putida is a bacterium known for its metabolic versatility and ability to tolerate various environmental stresses . It has been engineered to utilize non-native sugars like D-xylose, demonstrating its adaptability through metabolic engineering and adaptive laboratory evolution (ALE) . Recombinant P. putida strains can co-utilize xylose with glucose and other lignocellulosic sugars and aromatics, making them attractive biocatalysts for producing various compounds, including polyhydroxyalkanoates, cis, cis-muconate, dicarboxylic acids, biosurfactants, and amino acids .

Role of PPA in Toluene Tolerance

P. putida DOT-T1E exhibits tolerance to toluene and other toxic hydrocarbons by extruding the toxic compounds from the cell using efflux pumps . Proteomic analysis has revealed that inorganic pyrophosphatase is involved in providing the necessary energetic support for these reactions, suggesting its role in the bacterium's response to the stress caused by toluene .

PPA as a Target for Metabolic Engineering

Metabolic engineering of P. putida strains often involves manipulating key enzymes to enhance specific metabolic pathways. For instance, researchers have derepressed native glycolysis by deleting the local transcriptional regulator gene hexR and enhanced the pentose phosphate pathway by introducing exogenous transketolase and transaldolase .

Application of Recombinant P. putida

Recombinant P. putida strains have been developed for various applications:

• Biocatalysis: P. putida can be used as a biocatalyst for the production of polyhydroxyalkanoates, cis, cis-muconate, and other valuable compounds from xylose or complex lignocellulosic hydrolysates .

• Biodegradation: P. putida strains can biodegrade organofluorine compounds at millimolar concentrations .

• Cell Surface Display: Lipase has been displayed on the cell surface of P. putida KT2442 using OprF as an anchoring motif, resulting in high whole-cell lipase activity and stability in organic solvents .

• PHA Accumulation: Manipulation of genes like fadD in P. putida can affect the accumulation and composition of polyhydroxyalkanoates (PHA), a type of bioplastic .

Examples of Research Findings

• Deletion of the fadD gene in P. putida CA-3 resulted in no detectable growth or PHA accumulation with 10-phenyldecanoic acid, decanoic acid, and longer-chain substrates . The complemented mutant regained 70% to 90% of the growth and PHA-accumulating ability of the wild-type strain, depending on the substrate .

• The whole-cell lipase activity of recombinant P. putida KT2442 harboring pMO188PL was more than fivefold higher than that of recombinant Escherichia coli displaying lipase in the same manner .

• Engineered P. putida strains have shown enhanced expression of transaldolase and xylose isomerase, along with derepressed glycolysis, as key events during the adaptation process to utilize the non-native carbon source xylose .

Potential Applications

Because of its metabolic versatility and adaptability, recombinant P. putida with engineered PPA and related metabolic pathways holds promise for a wide range of biotechnological applications, including:

• Biomanufacturing of chemicals and biofuels: By optimizing PPA activity and carbon flux, P. putida can be engineered to efficiently produce valuable compounds from renewable resources .

• Bioremediation of pollutants: P. putida strains can be engineered to degrade environmental pollutants, such as organofluorine compounds and hydrocarbons .

• Production of bioplastics: By manipulating PHA synthesis pathways, P. putida can be used to produce sustainable bioplastics with tailored properties .