Global changes in the proteome of Cupriavidus necator H16 during poly-(3-hydroxybutyrate) synthesis from various biodiesel by-product substrates

Additional file 1: Table S1. P-scores of proteomic runs of C. necator H16 grown with different substrates

Transcription levels of the two <i>P</i>. <i>putida</i> LS46 PHA Synthases under three experimental conditions ^a, as determined by RT-qPCR.

<p>^a Exponential versus stationary growth phase in waste glycerol versus exponential growth phase in waste free fatty acids</p><p>^b cDNA values of each gene from10 ng total cDNA input for RT-qPCR under three studied experimental conditions</p><p>^c Analysis of the qPCR data by a Two-tail T-test revealed that abundance of <i>pha</i>C1 transcripts in the WFA culture were significantly different from the abundance of <i>pha</i>C1 transcripts in theWG cultures (P = 0.04, where the threshold was P = 0.05), whereas all the others were not significant from each other.</p><p>Transcription levels of the two <i>P</i>. <i>putida</i> LS46 PHA Synthases under three experimental conditions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142322#t002fn001" target="_blank">^a</a>, as determined by RT-qPCR.</p

Expression values of genes and gene products involved in proposed mcl-PHA metabolism derived from waste glycerol (WG) culture of <i>P</i>. <i>putida</i> LS46.

<p>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.</p

Quantitative ‘Omics Analyses of Medium Chain Length Polyhydroxyalkanaote Metabolism in <i>Pseudomonas putida</i> LS46 Cultured with Waste Glycerol and Waste Fatty Acids

<div><p>Transcriptomes and proteomes of <i>Pseudomonas putida</i> 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 <i>P</i>. <i>putida</i> LS46. Active mcl-PHA synthesis by <i>P</i>. <i>putida</i> 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 <i>P</i>. <i>putida</i> LS46 cells in different physiological states, when cultured on the two waste carbon sources. Optimal polymer production by <i>P</i>. <i>putida</i> 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 <i>P</i>. <i>putida</i> LS46 using biodiesel waste streams.</p></div

Growth of <i>P</i>. <i>putida</i> LS46 cultured with waste glycerol (WG) and waste fatty acids (WFA).

<p><i>P</i>. <i>putida</i> LS46 was cultured in Ramsay’s Minimal Medium containing A) WG and B) WFA. Solid triangles, Biomass; Solid black circles, mcl-PHA synthesis; Solid gray circles, glycerol concentrations (A) and fatty acid concentration (B); Open circles, nitrogen concentrations. Error bars represent standard deviations about the means calculated from three independent, biological replicate experiments. Arrows indicate samples taken for subsequent RNA and Protein isolation.</p