Transcriptome remodeling of <i>Pseudomonas putida</i> KT2440 during mcl-PHAs synthesis: effect of different carbon sources and response to nitrogen stress

Możejko-Ciesielska, Justyna; Pokój, Tomasz; Ciesielski, Sławomir

doi:10.1007/s10295-018-2042-4

Cited by 29 publications

(19 citation statements)

References 50 publications

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“…The production of ACC proteins was repressed when the bacteria entered the stationary phase. These data are consistent with our previous RNA-seq analysis that the transcripts levels of genes coding for acetyl-CoA carboxylase were depended on the growth phase of P. putida KT2440 and their overexpression may influence on the rate of polyhydroxyalkanoates biosynthesis [13].…”

Section: Resultssupporting

confidence: 92%

A 2D-DIGE-based proteomic analysis brings new insights into cellular responses of Pseudomonas putida KT2440 during polyhydroxyalkanoates synthesis

Możejko-Ciesielska

Mostek

2019

Microb Cell Fact

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Background 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. 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. Results The 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. Conclusions The 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. Electronic supplementary material The online version of this article (10.1186/s12934-019-1146-5) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 92%

A 2D-DIGE-based proteomic analysis brings new insights into cellular responses of Pseudomonas putida KT2440 during polyhydroxyalkanoates synthesis

Możejko-Ciesielska

Mostek

2019

Microb Cell Fact

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“…Then, its repression was observed at 48 h when mcl-PHAs were not synthesized, indicating that this protein could affect the intracellular biopolymers' accumulation. These data are consistent with previous RNA-seq analysis, confirming that genes related to the ED pathway could be potential targets in improving mcl-PHAs synthesis [8].…”

Section: Carbon and Energy Metabolismsupporting

confidence: 92%

“…PhaG encodes transacylase, which is not co-localized with the PHA biosynthesis gene cluster [7]. Możejko-Ciesielska et al [8] suggested that the phaG gene could be associated with the mcl-PHAs synthesis process not only on non-related carbon sources but also on related ones.…”

Section: Introductionmentioning

confidence: 99%

Proteomic Response of Pseudomonas putida KT2440 to Dual Carbon-Phosphorus Limitation during mcl-PHAs Synthesis

Możejko-Ciesielska

Serafim

2019

Biomolecules

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Pseudomonas putida KT2440, one of the best characterized pseudomonads, is a metabolically versatile producer of medium-chain-length polyhydroxyalkanoates (mcl-PHAs) that serves as a model bacterium for molecular studies. The synthesis of mcl-PHAs is of great interest due to their commercial potential. Carbon and phosphorus are the essential nutrients for growth and their limitation can trigger mcl-PHAs’ production in microorganisms. However, the specific molecular mechanisms that drive this synthesis in Pseudomonas species under unfavorable growth conditions remain poorly understood. Therefore, the proteomic responses of Pseudomonas putida KT2440 to the limited carbon and phosphorus levels in the different growth phases during mcl-PHAs synthesis were investigated. The data indicated that biopolymers’ production was associated with the cell growth of P. putida KT2440 under carbon- and phosphorus-limiting conditions. The protein expression pattern changed during mcl-PHAs synthesis and accumulation, and during the different physiological states of the microorganism. The data suggested that the majority of metabolic activities ceased under carbon and phosphorus limitation. The abundance of polyhydroxyalkanoate granule-associated protein (PhaF) involved in PHA synthesis increased significantly at 24 and 48 h of the cultivations. The activation of proteins belonging to the phosphate regulon was also detected. Moreover, these results indicated changes in the protein profiles related to amino acids metabolism, replication, transcription, translation, stress response mechanisms, transport or signal transduction. The presented data allowed the investigation of time-course proteome alterations in response to carbon and phosphorus limitation, and PHAs synthesis. This study provided information about proteins that can be potential targets in improving the efficiency of mcl-PHAs synthesis.

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“…The bulk of production occurred in stationary phase, ~12 h after carbon depletion. Production in late exponential or stationary phase is typical for several products in P. putida 29 , 30 . To test the use of galactose as a carbon source, we also engineered a galactose utilization strain via genomic integration of a galETKM operon 25 , 26 and here production of indigoidine was negligible (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Genome-scale metabolic rewiring improves titers rates and yields of the non-native product indigoidine at scale

et al. 2020

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High titer, rate, yield (TRY), and scalability are challenging metrics to achieve due to trade-offs between carbon use for growth and production. To achieve these metrics, we take the minimal cut set (MCS) approach that predicts metabolic reactions for elimination to couple metabolite production strongly with growth. We compute MCS solution-sets for a non-native product indigoidine, a sustainable pigment, in Pseudomonas putida KT2440, an emerging industrial microbe. From the 63 solution-sets, our omics guided process identifies one experimentally feasible solution requiring 14 simultaneous reaction interventions. We implement a total of 14 genes knockdowns using multiplex-CRISPRi. MCS-based solution shifts production from stationary to exponential phase. We achieve 25.6 g/L, 0.22 g/l/h, and ~50% maximum theoretical yield (0.33 g indigoidine/g glucose). These phenotypes are maintained from batch to fed-batch mode, and across scales (100-ml shake flasks, 250-ml ambr®, and 2-L bioreactors).

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Transcriptome remodeling of Pseudomonas putida KT2440 during mcl-PHAs synthesis: effect of different carbon sources and response to nitrogen stress

Cited by 29 publications

References 50 publications

A 2D-DIGE-based proteomic analysis brings new insights into cellular responses of Pseudomonas putida KT2440 during polyhydroxyalkanoates synthesis

A 2D-DIGE-based proteomic analysis brings new insights into cellular responses of Pseudomonas putida KT2440 during polyhydroxyalkanoates synthesis

Proteomic Response of Pseudomonas putida KT2440 to Dual Carbon-Phosphorus Limitation during mcl-PHAs Synthesis

Genome-scale metabolic rewiring improves titers rates and yields of the non-native product indigoidine at scale

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