Abstract
BackgroundPolyhydroxyalkanoates (PHAs) have attracted much attention in recent years as natural alternatives to petroleum-based synthetic polymers that can be broadly used in many applications. Pseudomonas putida KT2440 is a metabolically versatile microorganism that is able to synthesize medium-chain-length PHAs (mcl-PHAs). The phenomena that drive mcl-PHAs synthesis and accumulation seems to be complex and are still poorly understood. Therefore, here we determine new insights into cellular responses of Pseudomonas putida KT2440 during biopolymers production using two-dimensional difference gel-electrophoresis (2D-DIGE) followed by MALDI TOF/TOF mass spectrometry.ResultsThe maximum mcl-PHAs content in Pseudomonas putida KT2440 cells was 24% of cell dry weight (CDW) and was triggered by nitrogen depletion. Proteomic analysis allowed the detection of 150 and 131 protein spots differentially regulated at 24 h and 48 h relative to the cell growth stage (8 h), respectively. From those, we successfully identified 84 proteins that had altered expression at 24 h and 74 proteins at 48 h of the mcl-PHAs synthesis process. The protein–protein interactions network indicated that the majority of identified proteins were functionally linkage. The abundance of proteins involved in carbon metabolism were significantly decreased at 24 h and 48 h of the cultivations. Moreover, proteins associated with ATP synthesis were up-regulated suggesting that the enhanced energy metabolism was necessary for the mcl-PHAs accumulation. Furthermore, the induction of proteins involved in nitrogen metabolism, ribosome synthesis and transport was observed. Our results indicate that mcl-PHAs accumulated in the bacterial cells changed the protein abundance involved in stress response and cellular homeostasis.ConclusionsThe presented data allow us to investigate time-course proteome rearrangement in response to nitrogen limitation and biopolyesters accumulation. Our results have pointed out novel proteins that might take part in cellular responses of mcl-PHA-accumulated bacteria. The study provides an additional knowledge that could be helpful to improve the efficiency of the bioprocess and make it more economically feasible.
Highlights
Polyhydroxyalkanoates (PHAs) have attracted much attention in recent years as natural alternatives to petroleum-based synthetic polymers that can be broadly used in many applications
The key genes involved in the mcl-PHAs biosynthesis are known, there is a lack of information about the regulation mechanisms that are responsible for this process
This study presents a proteomic analysis of mcl-PHAs synthesis by Pseudomonas putida KT440 grown on sodium gluconate under nitrogen deficiency conditions
Summary
Polyhydroxyalkanoates (PHAs) have attracted much attention in recent years as natural alternatives to petroleum-based synthetic polymers that can be broadly used in many applications. Since the publication of Pseudomonas putida KT2440 complete genome, it has been considered as a model microorganism for genetic studies [3, 4] It allowed to analyze the ability of this bacterium to survive under stressful conditions and to synthesize biopolyesters, our knowledge about its metabolism at the molecular level has increased significantly. PHAs metabolism is encoded by the pha cluster and may be driven by the network of local and global regulators controlling the pathways involved in carbon and nitrogen assimilation [5] It is not yet clear how Pseudomonas putida KT2440 re-arranges its whole metabolism under nutrients starvation and the excess of related substrates, resulting in a high level of PHAs synthesis and accumulation. There is still a lack of studies that considered the cellular regulatory mechanisms leading to biopolyesters production from related carbon sources using time-course proteomic approach
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