Project description:Transcriptomics analysis of biopolymer (medium chain length polyhydroxyalkanoate) producing strain P.putida LS46 cultured with biodiesel derived waste carbon sources: studies of cellular adaptation to the industrial waste streams and metabolic profiling under the polymer producing conditions. We are reporting RNAseq analysis data here as part of our multi-level Omics study of medium chain length polyhydroxyalkanoate (mcl-PHA) producing strain P.putida LS46 culture with biodiesel derived waste glycerol and waste fatty acids. The data presented here will be used in two separate manuscripts. The objectives of this study are a): to evaluate cellular responses of P.putida LS46 under industrial waste stream. b): to study gene expression profile under two selected mcl-PHA producing conditions of P.putida LS46. Comparative multi-level Omics study: for objective a): Exponential P.putida LS46 cell from waste glycerol culture compared against reagent grade pure glycerol culture. For objective b): Two mcl-PHA producing conditions, namely stationary phase waste glycerol culture and exponential phase waste fatty acid culture of P.putida LS46, were compared against exponential phase waste glycerol culture of P.putida LS46. Major results from objective a): The waste glycerol substrate induced expression of a large number of genes putatively involved in heavy metal tolerance, including three gene clusters: a putative cusABC transcript unit and two copies of copAB, which are usually involved in copper resistance and tolerance to other monovalent heavy metals. A local gene relocation was observed in cluster 1 consisting cusABC and copAB relative to the KT2440 type strain according to the phylogenetic and gene neighbourhood analyses on various P. putida strains. P. putida LS46 also contains 11 putative MerR family regulators, which sense various environmental stimuli including heavy metals. MerR-1 is an ortholog of the copper response regulator of other gram-negative bacteria, and was highly up-regulated in waste glycerol cultures. Finally, a number of genes involved in cell responses to high extra-cellular Na+ concentrations, and genes of the fatty acid beta-oxidation pathway were up-regulated in waste glycerol cultures Major results from objective b): Regardless to the type of substrates, up-regulation of two mcl-PHA synthase (PhaC1 and PhaC2), and two phasin proteins (PhaF and PhaI) are the most common genotype under mcl-PHA production conditions. PhaG and possible PhaJ4 connect fatty acid de novo synthesis to mcl-PHA in waste glycerol culture. Interestingly, expression of gene, fabZ, in production of unsaturated fatty acid from fatty acid de novo synthesis was only observed in waste glycerol culture. On the other hand, PhaJ1 and PhaJ4 derived mcl-PHA production via fatty acid beta-oxidation was observed under waste fatty acid culture. These results would help to explain observed different production kinetics and monomer distribution of the polymer. Although under active mcl-PHA production condition, depression on the expression of glpF genes in glycerol transportation system prevent further channelling extra-cellular glycerol into the cell. Waste glycerol culture also triggers trahalose synthesis pathway, a potential competing pathway during mcl-PHA synthesizing. In waste fatty acid culture, the intermediates (acyl-CoA and 3-hydroxyacyl-CoA) of fatty acid beta-oxidation were used for mcl-PHA production and were also likely hydrolysed to their free acid forms via an up-regulated thioesteras coding gene, tesA. Acetyl-CoA cleaved from the pathway was clearly channeled into glyoxylate shut for C2 carbon assimilation over spillage as CO2 through TCA cycle or used in fatty acid biosynthesis pathway. In total 4 sampling points, namely exponential phase of pure glycerol, waste glycerol and waste free fatty acids cultures, and stationary phase of waste glycerol culture. For each sampling point, 2 biological replicates were taken. (Thus 8 samples in total)

Project description:Transcriptomics analysis of biopolymer (medium chain length polyhydroxyalkanoate) producing strain P.putida LS46 cultured with biodiesel derived waste carbon sources: studies of cellular adaptation to the industrial waste streams and metabolic profiling under the polymer producing conditions. We are reporting RNAseq analysis data here as part of our multi-level Omics study of medium chain length polyhydroxyalkanoate (mcl-PHA) producing strain P.putida LS46 culture with biodiesel derived waste glycerol and waste fatty acids. The data presented here will be used in two separate manuscripts. The objectives of this study are a): to evaluate cellular responses of P.putida LS46 under industrial waste stream. b): to study gene expression profile under two selected mcl-PHA producing conditions of P.putida LS46. Comparative multi-level Omics study: for objective a): Exponential P.putida LS46 cell from waste glycerol culture compared against reagent grade pure glycerol culture. For objective b): Two mcl-PHA producing conditions, namely stationary phase waste glycerol culture and exponential phase waste fatty acid culture of P.putida LS46, were compared against exponential phase waste glycerol culture of P.putida LS46. Major results from objective a): The waste glycerol substrate induced expression of a large number of genes putatively involved in heavy metal tolerance, including three gene clusters: a putative cusABC transcript unit and two copies of copAB, which are usually involved in copper resistance and tolerance to other monovalent heavy metals. A local gene relocation was observed in cluster 1 consisting cusABC and copAB relative to the KT2440 type strain according to the phylogenetic and gene neighbourhood analyses on various P. putida strains. P. putida LS46 also contains 11 putative MerR family regulators, which sense various environmental stimuli including heavy metals. MerR-1 is an ortholog of the copper response regulator of other gram-negative bacteria, and was highly up-regulated in waste glycerol cultures. Finally, a number of genes involved in cell responses to high extra-cellular Na+ concentrations, and genes of the fatty acid beta-oxidation pathway were up-regulated in waste glycerol cultures Major results from objective b): Regardless to the type of substrates, up-regulation of two mcl-PHA synthase (PhaC1 and PhaC2), and two phasin proteins (PhaF and PhaI) are the most common genotype under mcl-PHA production conditions. PhaG and possible PhaJ4 connect fatty acid de novo synthesis to mcl-PHA in waste glycerol culture. Interestingly, expression of gene, fabZ, in production of unsaturated fatty acid from fatty acid de novo synthesis was only observed in waste glycerol culture. On the other hand, PhaJ1 and PhaJ4 derived mcl-PHA production via fatty acid beta-oxidation was observed under waste fatty acid culture. These results would help to explain observed different production kinetics and monomer distribution of the polymer. Although under active mcl-PHA production condition, depression on the expression of glpF genes in glycerol transportation system prevent further channelling extra-cellular glycerol into the cell. Waste glycerol culture also triggers trahalose synthesis pathway, a potential competing pathway during mcl-PHA synthesizing. In waste fatty acid culture, the intermediates (acyl-CoA and 3-hydroxyacyl-CoA) of fatty acid beta-oxidation were used for mcl-PHA production and were also likely hydrolysed to their free acid forms via an up-regulated thioesteras coding gene, tesA. Acetyl-CoA cleaved from the pathway was clearly channeled into glyoxylate shut for C2 carbon assimilation over spillage as CO2 through TCA cycle or used in fatty acid biosynthesis pathway.

Project description:Sohn2010 - Genome-scale metabolic network of Pseudomonas putida (PpuMBEL1071) This model is described in the article: In silico genome-scale metabolic analysis of Pseudomonas putida KT2440 for polyhydroxyalkanoate synthesis, degradation of aromatics and anaerobic survival. Sohn SB, Kim TY, Park JM, Lee SY. Biotechnol J 2010 Jul; 5(7): 739-750 Abstract: Genome-scale metabolic models have been appearing with increasing frequency and have been employed in a wide range of biotechnological applications as well as in biological studies. With the metabolic model as a platform, engineering strategies have become more systematic and focused, unlike the random shotgun approach used in the past. Here we present the genome-scale metabolic model of the versatile Gram-negative bacterium Pseudomonas putida, which has gained widespread interest for various biotechnological applications. With the construction of the genome-scale metabolic model of P. putida KT2440, PpuMBEL1071, we investigated various characteristics of P. putida, such as its capacity for synthesizing polyhydroxyalkanoates (PHA) and degrading aromatics. Although P. putida has been characterized as a strict aerobic bacterium, the physiological characteristics required to achieve anaerobic survival were investigated. Through analysis of PpuMBEL1071, extended survival of P. putida under anaerobic stress was achieved by introducing the ackA gene from Pseudomonas aeruginosa and Escherichia coli. This model is hosted on BioModels Database and identified by: MODEL1507180043. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

			Action	DRS
	2021_12_06_SBBS_JLT_10A-4_S1-G10_1_2442.zip	Other
	2021_12_06_SBBS_JLT_10A-E_S1-C4_1_2388.zip	Other
	2021_12_06_SBBS_JLT_10B-4_S1-G11_1_2443.zip	Other
	2021_12_06_SBBS_JLT_10B-E_S1-C5_1_2389.zip	Other
	2021_12_06_SBBS_JLT_10C-4_S1-G12_1_2444.zip	Other

Dataset Information

The effect of multiple phaJ deletions Pseudomonas putida KT2440 on polyhydroxyalkanoates synthesis (part2)

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Publications

β-oxidation-polyhydroxyalkanoates synthesis relationship in Pseudomonas putida KT2440 revisited.

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