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Quantitative 'Omics Analyses of Medium Chain Length Polyhydroxyalkanaote Metabolism in Pseudomonas putida LS46 Cultured with Waste Glycerol and Waste Fatty Acids.

Fu J, Sharma P, Spicer V, Krokhin OV, Zhang X, Fristensky B, Cicek N, Sparling R, Levin DB - PLoS ONE (2015)

Bottom Line: Active mcl-PHA synthesis by P. putida LS46 was associated with high expression levels of key mcl-PHA biosynthesis genes and/or gene products including monomer-supplying proteins, PHA synthases, and granule-associated proteins. 'Omics data suggested that expression of these genes were regulated by different genetic mechanisms in P. putida LS46 cells in different physiological states, when cultured on the two waste carbon sources.Mapping the 'Omics data to the mcl-PHA biosynthetic pathway revealed significant variations in gene expression, primarily involved in: 1) glycerol transportation; 2) enzymatic reactions that recycle reducing equivalents and produce key mcl-PHA biosynthesis pathway intermediates (e.g. NADH/NADPH, acetyl-CoA).Understanding the relationships between genome content, gene and gene product expression, and how these factors influence polymer synthesis, will aid in optimization of mcl-PHA production by P. putida LS46 using biodiesel waste streams.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosystem Engineering, University of Manitoba, Winnipeg, Manitoba, Canada.

ABSTRACT
Transcriptomes and proteomes of Pseudomonas putida LS46 cultured with biodiesel-derived waste glycerol or waste free fatty acids, as sole carbon sources, were compared under conditions that were either permissive or non-permissive for synthesis of medium chain length polyhydroxyalkanoates (mcl-PHA). The objectives of this study were to elucidate mechanisms that influence activation of biopolymer synthesis, intra-cellular accumulation, and monomer composition, and determine if these were physiologically specific to the carbon sources used for growth of P. putida LS46. Active mcl-PHA synthesis by P. putida LS46 was associated with high expression levels of key mcl-PHA biosynthesis genes and/or gene products including monomer-supplying proteins, PHA synthases, and granule-associated proteins. 'Omics data suggested that expression of these genes were regulated by different genetic mechanisms in P. putida LS46 cells in different physiological states, when cultured on the two waste carbon sources. Optimal polymer production by P. putida LS46 was primarily limited by less efficient glycerol metabolism during mcl-PHA synthesis on waste glycerol. Mapping the 'Omics data to the mcl-PHA biosynthetic pathway revealed significant variations in gene expression, primarily involved in: 1) glycerol transportation; 2) enzymatic reactions that recycle reducing equivalents and produce key mcl-PHA biosynthesis pathway intermediates (e.g. NADH/NADPH, acetyl-CoA). Active synthesis of mcl-PHAs was observed during exponential phase in cultures with waste free fatty acids, and was associated with the fatty acid beta-oxidation pathway. A putative Thioesterase in the beta-oxidation pathway that may regulate the level of fatty acid beta-oxidation intermediates, and thus carbon flux to mcl-PHA biosynthesis, was highly up-regulated. Finally, the data suggested that differences in expression of selected fatty acid metabolism and mcl-PHA monomer-supplying enzymes may play a role in determining the monomer composition of mcl-PHA polymers. Understanding the relationships between genome content, gene and gene product expression, and how these factors influence polymer synthesis, will aid in optimization of mcl-PHA production by P. putida LS46 using biodiesel waste streams.

No MeSH data available.


Related in: MedlinePlus

Expression values of genes and gene products involved in proposed mcl-PHA metabolism derived from waste glycerol (WG) culture of P. putida LS46.Numbers in each column (from left to right) represent: the gene locus tag; RNA abundance during Exponential phase of WG cultures; Protein abundance during Exponential phase of WG cultures; Rnet and Pnet values from WG cultures in Stationary phase versus Exponential phase; Significantly up-regulated mRNAs or proteins are indicated in green font; Significantly down-regulated mRNA or proteins are indicated in red font; -, not detected, and therefore no Rnet or Pnet value associated. A star symbol in front of gene locus tag indicates proteins (if detected) with S/N (signal to noise) ratio less than 2.8, and thus having FDR greater than 10%. USFA: unsaturated fatty acid. Gene symbols for each putative pathway protein were also given. IDH1: putative NAD+-dependent isocitrate dehydrogenase; IDH2: putative NADP+-dependent isocitrate dehydrogenase; PDC: Pyruvate dehydrogenase complex; 6PGD: 6-phosphogluconate dehydrogenase.
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pone.0142322.g002: Expression values of genes and gene products involved in proposed mcl-PHA metabolism derived from waste glycerol (WG) culture of P. putida LS46.Numbers in each column (from left to right) represent: the gene locus tag; RNA abundance during Exponential phase of WG cultures; Protein abundance during Exponential phase of WG cultures; Rnet and Pnet values from WG cultures in Stationary phase versus Exponential phase; Significantly up-regulated mRNAs or proteins are indicated in green font; Significantly down-regulated mRNA or proteins are indicated in red font; -, not detected, and therefore no Rnet or Pnet value associated. A star symbol in front of gene locus tag indicates proteins (if detected) with S/N (signal to noise) ratio less than 2.8, and thus having FDR greater than 10%. USFA: unsaturated fatty acid. Gene symbols for each putative pathway protein were also given. IDH1: putative NAD+-dependent isocitrate dehydrogenase; IDH2: putative NADP+-dependent isocitrate dehydrogenase; PDC: Pyruvate dehydrogenase complex; 6PGD: 6-phosphogluconate dehydrogenase.

Mentions: The substrate (glycerol) consumption rate of P. putida LS46 in WG cultures was low once the cells reached stationary phase, compared to the substrate (free fatty acids) consumption rate in WFA cultures, under active mcl-PHA biosynthesis conditions (Fig 1A). This was associated with significant reduction in protein expression of glpF gene, a transmembrane glycerol transportation facilitator, in the proteome of P. putida LS46 (Fig 2) suggesting the efficient transportation of extra-cellular glycerol into cytoplasm was restrained during stationary phase. While expression of other essential genes and homologs (i.e. oprB porin homologs) for glycerol transportation and catabolic initiation did not changed significantly.


Quantitative 'Omics Analyses of Medium Chain Length Polyhydroxyalkanaote Metabolism in Pseudomonas putida LS46 Cultured with Waste Glycerol and Waste Fatty Acids.

Fu J, Sharma P, Spicer V, Krokhin OV, Zhang X, Fristensky B, Cicek N, Sparling R, Levin DB - PLoS ONE (2015)

Expression values of genes and gene products involved in proposed mcl-PHA metabolism derived from waste glycerol (WG) culture of P. putida LS46.Numbers in each column (from left to right) represent: the gene locus tag; RNA abundance during Exponential phase of WG cultures; Protein abundance during Exponential phase of WG cultures; Rnet and Pnet values from WG cultures in Stationary phase versus Exponential phase; Significantly up-regulated mRNAs or proteins are indicated in green font; Significantly down-regulated mRNA or proteins are indicated in red font; -, not detected, and therefore no Rnet or Pnet value associated. A star symbol in front of gene locus tag indicates proteins (if detected) with S/N (signal to noise) ratio less than 2.8, and thus having FDR greater than 10%. USFA: unsaturated fatty acid. Gene symbols for each putative pathway protein were also given. IDH1: putative NAD+-dependent isocitrate dehydrogenase; IDH2: putative NADP+-dependent isocitrate dehydrogenase; PDC: Pyruvate dehydrogenase complex; 6PGD: 6-phosphogluconate dehydrogenase.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4636370&req=5

pone.0142322.g002: Expression values of genes and gene products involved in proposed mcl-PHA metabolism derived from waste glycerol (WG) culture of P. putida LS46.Numbers in each column (from left to right) represent: the gene locus tag; RNA abundance during Exponential phase of WG cultures; Protein abundance during Exponential phase of WG cultures; Rnet and Pnet values from WG cultures in Stationary phase versus Exponential phase; Significantly up-regulated mRNAs or proteins are indicated in green font; Significantly down-regulated mRNA or proteins are indicated in red font; -, not detected, and therefore no Rnet or Pnet value associated. A star symbol in front of gene locus tag indicates proteins (if detected) with S/N (signal to noise) ratio less than 2.8, and thus having FDR greater than 10%. USFA: unsaturated fatty acid. Gene symbols for each putative pathway protein were also given. IDH1: putative NAD+-dependent isocitrate dehydrogenase; IDH2: putative NADP+-dependent isocitrate dehydrogenase; PDC: Pyruvate dehydrogenase complex; 6PGD: 6-phosphogluconate dehydrogenase.
Mentions: The substrate (glycerol) consumption rate of P. putida LS46 in WG cultures was low once the cells reached stationary phase, compared to the substrate (free fatty acids) consumption rate in WFA cultures, under active mcl-PHA biosynthesis conditions (Fig 1A). This was associated with significant reduction in protein expression of glpF gene, a transmembrane glycerol transportation facilitator, in the proteome of P. putida LS46 (Fig 2) suggesting the efficient transportation of extra-cellular glycerol into cytoplasm was restrained during stationary phase. While expression of other essential genes and homologs (i.e. oprB porin homologs) for glycerol transportation and catabolic initiation did not changed significantly.

Bottom Line: Active mcl-PHA synthesis by P. putida LS46 was associated with high expression levels of key mcl-PHA biosynthesis genes and/or gene products including monomer-supplying proteins, PHA synthases, and granule-associated proteins. 'Omics data suggested that expression of these genes were regulated by different genetic mechanisms in P. putida LS46 cells in different physiological states, when cultured on the two waste carbon sources.Mapping the 'Omics data to the mcl-PHA biosynthetic pathway revealed significant variations in gene expression, primarily involved in: 1) glycerol transportation; 2) enzymatic reactions that recycle reducing equivalents and produce key mcl-PHA biosynthesis pathway intermediates (e.g. NADH/NADPH, acetyl-CoA).Understanding the relationships between genome content, gene and gene product expression, and how these factors influence polymer synthesis, will aid in optimization of mcl-PHA production by P. putida LS46 using biodiesel waste streams.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosystem Engineering, University of Manitoba, Winnipeg, Manitoba, Canada.

ABSTRACT
Transcriptomes and proteomes of Pseudomonas putida LS46 cultured with biodiesel-derived waste glycerol or waste free fatty acids, as sole carbon sources, were compared under conditions that were either permissive or non-permissive for synthesis of medium chain length polyhydroxyalkanoates (mcl-PHA). The objectives of this study were to elucidate mechanisms that influence activation of biopolymer synthesis, intra-cellular accumulation, and monomer composition, and determine if these were physiologically specific to the carbon sources used for growth of P. putida LS46. Active mcl-PHA synthesis by P. putida LS46 was associated with high expression levels of key mcl-PHA biosynthesis genes and/or gene products including monomer-supplying proteins, PHA synthases, and granule-associated proteins. 'Omics data suggested that expression of these genes were regulated by different genetic mechanisms in P. putida LS46 cells in different physiological states, when cultured on the two waste carbon sources. Optimal polymer production by P. putida LS46 was primarily limited by less efficient glycerol metabolism during mcl-PHA synthesis on waste glycerol. Mapping the 'Omics data to the mcl-PHA biosynthetic pathway revealed significant variations in gene expression, primarily involved in: 1) glycerol transportation; 2) enzymatic reactions that recycle reducing equivalents and produce key mcl-PHA biosynthesis pathway intermediates (e.g. NADH/NADPH, acetyl-CoA). Active synthesis of mcl-PHAs was observed during exponential phase in cultures with waste free fatty acids, and was associated with the fatty acid beta-oxidation pathway. A putative Thioesterase in the beta-oxidation pathway that may regulate the level of fatty acid beta-oxidation intermediates, and thus carbon flux to mcl-PHA biosynthesis, was highly up-regulated. Finally, the data suggested that differences in expression of selected fatty acid metabolism and mcl-PHA monomer-supplying enzymes may play a role in determining the monomer composition of mcl-PHA polymers. Understanding the relationships between genome content, gene and gene product expression, and how these factors influence polymer synthesis, will aid in optimization of mcl-PHA production by P. putida LS46 using biodiesel waste streams.

No MeSH data available.


Related in: MedlinePlus