Project description:Fatty acid synthesis is closely linked to nutrient availability and cellular energetic status. The committed step in fatty acid synthesis is the acetyl CoA carboxylase. Eukaryotes have two genes encoding acetyl CoA carboxylases, one encoding a cytosolic enzyme and another coding for a mitochondrial enzyme. They catalyze the synthesis of malonyl CoA in the cytosol and the mitochondria, respectively. While cytosolic malonyl CoA is the precursor for fatty acid synthesis, mitochondrial malonyl CoA controls the transfer of fatty acyl group into the mitochondria by inhibiting carnitine/palmitoyl transferase activity and thus, regulates Î²-oxidation. In Saccharomyces cerevisiae, Î²-oxidation is restricted to the peroxisomes, raising the question of the function of the mitochondrial isoform (HFA1). In this study, we replaced the cytosolic Acc1 with Hfa1 expressed in the cytosol by removing the mitochondrial leader peptide, under control of the HFA1 promoter. We studied fatty acid synthesis and transcription profiles in this strain during starvation for carbon or nitrogen, using glucose or ethanol as the carbon source. Under all the conditions studied, the key sensor of energetic status, Snf1, was activated, indicating active inhibition of fatty acid synthesis. The pool size of fatty acids was smaller when Acc1 was replaced with truncated Hfa1 for fatty acid synthesis. Yet, the transcription profiles were similar in both the cases. These results point towards the conclusion that Hfa1 is either catalytically less efficient or it is more sensitive to inhibition by Snf1. Gene expression from a strain of Saccharomyces cerevisiae where cytosolic fatty acid synthesis occurs by mitochondrial acetyl CoA carboxylase (without its mitochondrial leader peptide) is compared with that in a reference strain while growing in chemostats on carbon or nitrogen starvation using glucose or ethanol as the carbon source. There are two strains (reference or mutant), two carbon sources (glucose or ethanol) and two limitations (carbon or nitrogen), resulting in 8 comparisons. Each array was performed in duplicate, resulting in 16 CEL files. Growth was limited by either carbon or nitrogen. When carbon was the limited nutrient, we tested growth on either glucose or ethanol (both using ammonium sulfate as the nitrogen source). When ammonium sulfate was limiting, we used either glucose or ethanol as the carbon source.

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.

Dataset Information

Berzins2022 - Crypthecodinium cohnii: glucose and glycerol uptake for production of DHA precursor acetyl-CoA

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