The synthesis and degradation of polyhydroxyalkanoates (PHAs), the storage polymer of many bacteria, is linked to the operation of central carbon metabolism. To rationalize the impact of PHA accumulation on central carbon metabolism of the prototype bacterium Pseudomonas putida, we have revisited PHA production in quantitative physiology experiments in the wild-type strain vs. a PHA negative mutant growing under low nitrogen conditions. When octanoic acid was used as PHA precursor and as carbon and energy source, we have detected higher intracellular flux via acetyl-CoA in the mutant strain than in the wild type, which correlates with the stimulation of the TCA cycle and glyoxylate shunt observed on the transcriptional level. The mutant defective in carbon and energy storage spills the additional resources, releasing CO(2) instead of generating biomass. Hence, P. putida operates the metabolic network to optimally exploit available resources and channels excess carbon and energy to storage via PHA, without compromising growth. These findings demonstrate that the PHA metabolism plays a critical role in synchronizing global metabolism to availability of resources in PHA-producing microorganisms.
"PHAs are key players in the metabolism of many bacteria, involved in cell homeostasis through different functional roles. As mentioned before, PHA accumulation allows cells to dispose of excess carbon and reducing equivalents and to have these resources readily available when needed for different cellular processes (Escapa et al., 2012). However, PHAs are not the only polymers accumulated by bacteria. "
[Show abstract][Hide abstract] ABSTRACT: Bacterial polyhydroxyalkanoates (PHAs) are isotactic polymers that play a critical role in central metabolism, as they act as dynamic reservoirs of carbon and reducing equivalents. These polymers have a number of technical applications since they exhibit thermoplastic and elastomeric properties, making them attractive as a replacement of oil-derived materials. PHAs are accumulated under conditions of nutritional imbalance (usually an excess of carbon source with respect to a limiting nutrient, such as nitrogen or phosphorus). The cycle of PHA synthesis and degradation has been recognized as an important physiological feature when these biochemical pathways were originally described, yet its role in bacterial processes as diverse as global regulation and cell survival is just starting to be appreciated in full. In the present revision, the complex regulation of PHA synthesis and degradation at the transcriptional, translational, and metabolic levels are explored by analyzing examples in natural producer bacteria, such as Pseudomonas species, as well as in recombinant Escherichia coli strains. The ecological role of PHAs, together with the interrelations with other polymers and extracellular substances, is also discussed, along with their importance in cell survival, resistance to several types of environmental stress, and planktonic-versus-biofilm lifestyle. Finally, bioremediation and plant growth promotion are presented as examples of environmental applications in which PHA accumulation has successfully been exploited.
"It was shown before that intracellular NADH concentrations are crucial for cell growth [41,42]. Similar observations of the importance of the NADH/NAD+ ratio for PHA synthesis from fatty acids in P. putida were published before [43-45]. "
[Show abstract][Hide abstract] ABSTRACT: Background
Pseudomnas putida is a natural producer of medium chain length polyhydroxyalkanoates (mcl-PHA), a polymeric precursor of bioplastics. A two-fold increase of mcl-PHA production via inactivation of the glucose dehydrogenase gene gcd, limiting the metabolic flux towards side products like gluconate was achieved before. Here, we investigated the overproduction of enzymes catalyzing limiting steps of mcl-PHA precursor formation.
A genome-based in silico model for P. putida KT2440 metabolism was employed to identify potential genetic targets to be engineered for the improvement of mcl-PHA production using glucose as sole carbon source. Here, overproduction of pyruvate dehydrogenase subunit AcoA in the P. putida KT2440 wild type and the Δgcd mutant strains led to an increase of PHA production. In controlled bioreactor batch fermentations PHA production was increased by 33% in the acoA overexpressing wild type and 121% in the acoA overexpressing Δgcd strain in comparison to P. putida KT2440. Overexpression of pgl-encoding 6-phosphoglucolactonase did not influence PHA production. Transcriptome analyses of engineered PHA producing P. putida in comparison to its parental strains revealed the induction of genes encoding glucose 6-phosphate dehydrogenase and pyruvate dehydrogenase. In addition, NADPH seems to be quantitatively consumed for efficient PHA synthesis, since a direct relationship between low levels of NADPH and high concentrations of the biopolymer were observed. In contrast, intracellular levels of NADH were found increased in PHA producing organisms.
Production of mcl-PHAs was enhanced in P. putida when grown on glucose via overproduction of a pyruvate dehydrogenase subunit (AcoA) in combination with a deletion of the glucose dehydrogenase (gcd) gene as predicted by in silico elementary flux mode analysis.
"A wide variety of microorganisms accumulate PHAs as a carbon and energy storage material in carbon-excess conditions when a major nutrient (typically nitrogen or phosphorus) is limiting (Elbahloul and Steinbüchel 2009; Rehm 2010; Brandl et al. 1988). Accumulation of excess carbon is a general mechanism used by Pseudomonas and is essential for resource balancing (de Eugenio et al. 2010a; Escapa et al. 2012). De Smet et al. (1983) detected the inclusion bodies in Pseudomonas olevorans for first time when grown on octane and identified is as polymer of 3-hydroxyoctanoate. "
[Show abstract][Hide abstract] ABSTRACT: A novel strain of Pseudomonas putida LS46 was isolated from wastewater on the basis of its ability to synthesize medium chain-length polyhydroxyalkanoates (mcl-PHAs). P.putida LS46 was differentiated from other P.putida strains on the basis of cpn60 (UT). The complete genome of P.putida LS46 was sequenced and annotated. Its chromosome is 5,86,2556 bp in size with GC ratio of 61.69. It is encoding 5316 genes, including 7 rRNA genes and 76 tRNA genes. Nucleotide sequence data of the complete P. putida LS46 genome was compared with nine other P. putida strains (KT2440, F1, BIRD-1, S16, ND6, DOT-T1E, UW4, W619 and GB-1) identified either as biocontrol agents or as bioremediation agents and isolated from different geographical region and different environment. BLASTn analysis of whole genome sequences of the ten P. putida strains revealed nucleotide sequence identities of 86.54 to 97.52%. P.putida genome arrangement was LS46 highly similar to P.putida BIRD1 and P.putida ND6 but was markedly different than P.putida DOT-T1E, P.putida UW4 and P.putida W619. Fatty acid biosynthesis (fab), fatty acid degradation (fad) and PHA synthesis genes were highly conserved among biocontrol and bioremediation P.putida strains. Six genes in pha operon of P. putida LS46 showed >98% homology at gene and proteins level. It appears that polyhydroxyalkanoate (PHA) synthesis is an intrinsic property of P. putida and was not affected by its geographic origin. However, all strains, including P. putida LS46, were different from one another on the basis of house keeping genes, and presence of plasmid, prophages, insertion sequence elements and genomic islands. While P. putida LS46 was not selected for plant growth promotion or bioremediation capacity, its genome also encoded genes for root colonization, pyoverdine synthesis, oxidative stress (present in other soil isolates), degradation of aromatic compounds, heavy metal resistance and nicotinic acid degradation, manganese (Mn II) oxidation. Genes for toluene or naphthalene degradation found in the genomes of P. putida F1, DOT-T1E, and ND6 were absent in the P. putida LS46 genome. Heavy metal resistant genes encoded by the P. putida W619 genome were also not present in the P. putida LS46 genome. Despite the overall similarity among genome of P.putida strains isolated for different applications and from different geographical location a number of differences were observed in genome arrangement, occurrence of transposon, genomic islands and prophage. It appears that P.putida strains had a common ancestor and by acquiring some specific genes by horizontal gene transfer it differed from other related strains.
AMB Express 06/2014; 4(1). DOI:10.1186/s13568-014-0037-8
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