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:Background. Transforming waste and non-food materials into bulk biofuels and chemicals represents a major stride in creating a sustainable bioindustry, optimizing the use of resources while reducing environmental footprints. Yet, despite these advancements, the production of high-value natural products often continues to rely on first-generation substrates, underscoring the intricate processes and specific requirements of their biosynthesis. This is also true for Streptomyces lividans, a renowned host organism celebrated for its capacity to produce and uncover a wide array of natural products, attributed to its genetic versatility and potent secondary metabolism. Given this context, it becomes imperative to assess and optimize this microorganism for the synthesis of natural products specifically from waste and non-food substrates. Results. We metabolically engineered S. lividans TK24 to heterologously produce the ribosomally synthesized and post-translationally modified peptide, bottromycin, as well as the polyketide, pamamycin. The modified strains successfully produced these compounds using waste and non-food model substrates like protocatechuate (derived from lignin), 4-hydroxybenzoate (sourced from plastic waste), and mannitol (from seaweed). Comprehensive transcriptomic and metabolomic analyses offered insights into how these substrates influenced the cellular metabolism of S. lividans. When evaluating production efficiency, S. lividans showcased remarkable tolerance, especially in a fed-batch process using a mineral medium containing the toxic aromatic 4-hydroxybenzoate, leading to enhanced and highly selective bottromycin production. Additionally, it generated a unique spectrum of pamamycins when cultured in mannitol-rich seaweed extract without the need for added nutrients. Conclusion. Our study showcases the successful production of high-value natural products using varied waste and non-food raw materials, thereby circumventing the reliance on costly, food-competing resources. S. lividans exhibited remarkable adaptability and resilience across these diverse substrates. When cultured on aromatic compounds, it displayed a distinct array of intracellular CoA esters, presenting promising avenues for polyketide production. Future research could focus on enhancing S. lividans' substrate utilization pathways to more efficiently process the intricate mixtures commonly found in waste and non-food sources.

Project description:Oxidative stress due to endogenous hydrogen peroxide production by Lactobacillus species is a well-known issue in the food industry. In this study, the transcriptional response to oxygen of Lactobacillus johnsonii, one of the H2O2-producing strains used in the food industry, was analyzed. It was found that aerobic growth conditions led to a more than two-fold downregulation of 45 genes as compared to anaerobic growth, whereas 6 genes were more than twofold upregulated. Among the upregulated genes were two genes that displayed significant homology to NADH-dependent oxidoreductase (NOX). The postulated transcriptional regulation of the nox promoter by oxygen was studied using a GUS-reporter construct, confirming a 2.1-fold upregulated GUS-expression upon aerobic growth. Exposure to sublethal levels of hydrogen peroxide did not result in significant regulation of the nox promoter. In a previous study of hydrogen peroxide production by L. johnsonii, a NADH flavin reductase (NFR) was identified to be involved in hydrogen peroxide production. An NFR-deficient derivative was strongly impaired in H2O2 production, but regained a partial H2O2 producing capacity upon prolonged oxygen exposure. The nox-promoter appeared to be 3.6-fold upregulated under aerobic conditions in the NFR-deficient background, which may imply a role of this gene in the regained H2O2 production. Indeed, deletion of the nox-gene in the NFR-deletion background, resulted in a strain that no longer produced H2O2, also during prolonged exposure to oxygen. The double-mutant (nfr, nox) displayed strongly impaired aerobic growth and oxygenation induced rapid growth stagnation that is not caused by H2O2. We conclude that H2O2 production in L. johnsonii is primarily dependent on NFR but can also involve an oxygen-inducible NADH oxidase under aerobic conditions. Moreover, our results imply that H2O2 production plays a prominent role in oxygen tolerance of L. johnsonii. loop design of the samples including two shortcuts

			Action	DRS
	DRR525860_1.fastq.gz	Fastqsanger.gz
	DRR525860_2.fastq.gz	Fastqsanger.gz
	DRR525861_1.fastq.gz	Fastqsanger.gz
	DRR525861_2.fastq.gz	Fastqsanger.gz
	DRR525862_1.fastq.gz	Fastqsanger.gz

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