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β-oxidation-polyhydroxyalkanoates synthesis relationship in Pseudomonas putida KT2440 revisited
Si Liu1,2, Tanja Narancic1,2, Jia-Lynn Tham1,2
1UCD Earth Institute and School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland.
Applied Microbiology and Biotechnology
|February 10, 2023
Summary
Pseudomonas putida KT2440 accumulates polyhydroxyalkanoates (PHA) through fatty acid metabolism. While PhaJ enzymes are key, other non-specific enzymes also contribute, revealing a redundant PHA synthesis pathway.
Area of Science:
- Microbial Biotechnology
- Metabolic Engineering
- Synthetic Biology
Background:
- Pseudomonas putida KT2440 is a model organism for medium-chain-length polyhydroxyalkanoate (mcl-PHA) accumulation.
- The enzyme (R)-specific enoyl-coenzyme A hydratase (PhaJ) is crucial for converting β-oxidation intermediates into PHA monomers using fatty acids.
- Three PhaJ homologues (PhaJ1, PhaJ4, MaoC) are present in P. putida KT2440, with PhaJ4 and PhaJ1 showing substrate preferences for C8-C10 and C6 fatty acids, respectively.
Purpose of the Study:
- To investigate the roles of PhaJ homologues in fatty acid metabolism and PHA biosynthesis in P. putida KT2440.
- To identify other potential enzymes involved in PHA monomer supply.
- To understand the redundancy and regulation of PHA synthesis pathways.
Main Methods:
- Construction and analysis of various P. putida KT2440 knockout mutants.
- Analysis of PHA content and monomer composition under different growth conditions.
- Proteome analysis to identify potential monomer-supplying enzymes.
Main Results:
- PhaJ4 was identified as the primary monomer supplier for PHA synthesis from fatty acids, with PhaJ1 contributing to shorter-chain fatty acids.
- Even after deleting all three PhaJ homologues, PHA accumulation persisted (up to 10.7% CDW).
- Deletion of PhaG, which links fatty acid and PHA synthesis, further reduced PHA content but did not abolish it.
- Proteome analysis suggested quinoprotein alcohol dehydrogenases (PedE, PedH) as potential suppliers, but their deletion did not eliminate PHA accumulation.
- PHA levels remained significant (2.2-14.8% CDW) even without known PhaJ or Ped enzymes, depending on fatty acid source and nitrogen availability.
Conclusions:
- PhaJ1, PhaJ4, and MaoC are important, but not the sole, contributors to PHA monomer synthesis from β-oxidation intermediates.
- The PHA synthesis pathway in P. putida KT2440 exhibits significant redundancy, with non-specific enzymes also supplying monomers.
- This highlights the robustness and adaptability of PHA metabolism in this model organism.
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