A novel programmable lysozyme-based lysis system in Pseudomonas putida for biopolymer production

Indexación: Scopus; Web of Science.Cell lysis is crucial for the microbial production of industrial fatty acids, proteins, biofuels, and biopolymers. In this work, we developed a novel programmable lysis system based on the heterologous expression of lysozyme. The inducible lytic system was tested in two Gram-negative bacterial strains, namely Escherichia coli and Pseudomonas putida KT2440. Before induction, the lytic system did not significantly arrest essential physiological parameters in the recombinant E. coli (ECPi) and P. putida (JBOi) strain such as specific growth rate and biomass yield under standard growth conditions. A different scenario was observed in the recombinant JBOi strain when subjected to PHA-producing conditions, where biomass production was reduced by 25% but the mcl-PHA content was maintained at about 30% of the cell dry weight. Importantly, the genetic construct worked well under PHA-producing conditions (nitrogen-limiting phase), where more than 95% of the cell population presented membrane disruption 16 h post induction, with 75% of the total synthesized biopolymer recovered at the end of the fermentation period. In conclusion, this new lysis system circumvents traditional, costly mechanical and enzymatic cell-disrupting procedures.https://www.nature.com/articles/s41598-017-04741-2.pd

Datasets for transcriptomics, q-proteomics and phenotype microarrays of polyphosphate metabolism mutants from Escherichia coli

Indexación: Scopus.Author acknowledges Fondecyt Grants 1120209, 1121170 and Anillo ACT-1107Here, we provide the dataset associated with our research article on the polyphosphate metabolism entitled, “Multi-level evaluation of Escherichia coli polyphosphate related mutants using global transcriptomic, proteomic and phenomic analyses”. By integrating different omics levels (transcriptome, proteome and phenome), we were able to study Escherichia coli polyphosphate mutant strains (Δppk1, Δppx, and Δppk1-ppx). We have compiled here all datasets from DNA microarrys, q-proteomic (Isotope-Coded Protein Labeling, ICPL) and phenomic (Phenotype microarray) raw data we have obtained in all polyP metabolism mutants.http://www.sciencedirect.com/science/article/pii/S2352340917300860?via%3Dihu

Biochemistry, genetics and biotechnology of glycerol utilization in Pseudomonas species

Indexación: Scopus.The use of renewable waste feedstocks is an environment-friendly choice contributing to the reduction of waste treatment costs and increasing the economic value of industrial by-products. Glycerol (1,2,3-propanetriol), a simple polyol compound widely distributed in biological systems, constitutes a prime example of a relatively cheap and readily available substrate to be used in bioprocesses. Extensively exploited as an ingredient in the food and pharmaceutical industries, glycerol is also the main by-product of biodiesel production, which has resulted in a progressive drop in substrate price over the years. Consequently, glycerol has become an attractive substrate in biotechnology, and several chemical commodities currently produced from petroleum have been shown to be obtained from this polyol using whole-cell biocatalysts with both wild-type and engineered bacterial strains. Pseudomonas species, endowed with a versatile and rich metabolism, have been adopted for the conversion of glycerol into value-added products (ranging from simple molecules to structurally complex biopolymers, e.g. polyhydroxyalkanoates), and a number of metabolic engineering strategies have been deployed to increase the number of applications of glycerol as a cost-effective substrate. The unique genetic and metabolic features of glycerol-grown Pseudomonas are presented in this review, along with relevant examples of bioprocesses based on this substrate – and the synthetic biology and metabolic engineering strategies implemented in bacteria of this genus aimed at glycerol valorization.https://sfamjournals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.1340

Editorial: Pathway, Genetic and Process Engineering of Microbes for Biopolymer Synthesis

Indexación ScopusTogether with the climate crisis, the heavy accumulation in oceans and soils of persistent pollutants, like polycyclic aromatics hydrocarbons and synthetic plastics, are the main drivers to impact nature and threaten human survival. It is particularly striking that most industrial polymers still originate from petrochemical sources—nearly 99% of the overall worldwide production. The result is materials that remain intact for centuries once deposited in the environment. Relying on non-renewable fossil chemicals limits our ability to establish a circular economy that promises to curb current emissions and contribute moderately to the global carbon cycle without surpassing its carrying capacity. For decades, commercial biopolymers have also been produced by microbial fermentation since nature has endowed many bacteria from urban sites to extreme environments (Orellana-Saez et al., 2019) with the enzymatic machinery to assemble these macromolecules. Despite the rapid pace of innovation, microbial biopolymers are still expensive to synthesize because the generally oxygen-intensive fermentation processes, downstream processing, and carbon feedstock cost boost production expenses (Oliveira et al., 2020). The biopolymers must additionally possess specific mechanical and physical properties to be processed industrially into products with a variety of applications (Moradali and Rehm, 2020).https://www.frontiersin.org/articles/10.3389/fbioe.2020.618383/ful

Fed-Batch mcl- Polyhydroxyalkanoates Production in Pseudomonas putida KT2440 and ΔphaZ Mutant on Biodiesel-Derived Crude Glycerol

Indexación ScopusCrude glycerol has emerged as a suitable feedstock for the biotechnological production of various industrial chemicals given its high surplus catalyzed by the biodiesel industry. Pseudomonas bacteria metabolize the polyol into several biopolymers, including alginate and medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHAs). Although P. putida is a suited platform to derive these polyoxoesters from crude glycerol, the attained concentrations in batch and fed-batch cultures are still low. In this study, we employed P. putida KT2440 and the hyper-PHA producer ΔphaZ mutant in two different fed-batch modes to synthesize mcl-PHAs from raw glycerol. Initially, the cells grew in a batch phase (μmax 0.21 h–1) for 22 h followed by a carbon-limiting exponential feeding, where the specific growth rate was set at 0.1 (h–1), resulting in a cell dry weight (CDW) of nearly 50 (g L–1) at 40 h cultivation. During the PHA production stage, we supplied the substrate at a constant rate of 50 (g h–1), where the KT2440 and the ΔphaZ produced 9.7 and 12.7 gPHA L–1, respectively, after 60 h cultivation. We next evaluated the PHA production ability of the P. putida strains using a DO-stat approach under nitrogen depletion. Citric acid was the main by-product secreted by the cells, accumulating in the culture broth up to 48 (g L–1) under nitrogen limitation. The mutant ΔphaZ amassed 38.9% of the CDW as mcl-PHA and exhibited a specific PHA volumetric productivity of 0.34 (g L–1 h–1), 48% higher than the parental KT2440 under the same growth conditions. The biosynthesized mcl-PHAs had average molecular weights ranging from 460 to 505 KDa and a polydispersity index (PDI) of 2.4–2.6. Here, we demonstrated that the DO-stat feeding approach in high cell density cultures enables the high yield production of mcl-PHA in P. putida strains using the industrial crude glycerol, where the fed-batch process selection is essential to exploit the superior biopolymer production hallmarks of engineered bacterial strains. © Copyright © 2021 Borrero-de Acuña, Rohde, Saldias and Poblete-Castro.https://www.frontiersin.org/articles/10.3389/fbioe.2021.642023/ful

Enhanced synthesis of medium-chain-length poly(3-hydroxyalkanoates) by inactivating the tricarboxylate transport system of Pseudomonas putida KT2440 and process development using waste vegetable oil

Indexación: Scopus.The use of waste materials as feedstock for biosynthesis of valuable compounds has been an intensive area of research aiming at diminishing the consumption of non-renewable materials. In this study, P. putida KT2440 was employed as a cell factory for the bioconversion of waste vegetable oil into medium-chain-length Polyhydroxyalkanoates. In the presence of the waste oil this environmental strain is capable of secreting enzymes with lipase activities that enhance the bioavailability of this hydrophobic carbon substrate. It was also found that the oxygen transfer coefficient is directly correlated with high PHA levels in KT2440 cells when metabolizing the waste frying oil. By knocking out the tctA gene, encoding for an enzyme of the tripartite carboxylate transport system, an enhanced intracellular level of mcl-PHA was found in the engineered strain when grown on fatty acids. Batch bioreactors showed that the KT2440 strain produced 1.01 (g⋅L −1 ) of PHA whereas the engineered ΔtctA P. putida strain synthesized 1.91 (g⋅L −1 ) after 72 h cultivation on 20 (g⋅L −1 ) of waste oil, resulting in a nearly 2-fold increment in the PHA volumetric productivity. Taken together, this work contributes to accelerate the pace of development for efficient bioconversion of waste vegetable oils into sustainable biopolymers. © 2018 Elsevier Ltd.https://www.sciencedirect.com/science/article/pii/S135951131831451X?via%3Dihu

In-Depth Genomic and Phenotypic Characterization of the Antarctic Psychrotolerant Strain Pseudomonas sp. MPC6 Reveals Unique Metabolic Features, Plasticity, and Biotechnological Potential

We obtained the complete genome sequence of the psychrotolerant extremophile Pseudomonas sp. MPC6, a natural Polyhydroxyalkanoates (PHAs) producing bacterium able to rapidly grow at low temperatures. Genomic and phenotypic analyses allowed us to situate this isolate inside the Pseudomonas fluorescens phylogroup of pseudomonads as well as to reveal its metabolic versatility and plasticity. The isolate possesses the gene machinery for metabolizing a variety of toxic aromatic compounds such as toluene, phenol, chloroaromatics, and TNT. In addition, it can use both C6- and C5-carbon sugars like xylose and arabinose as carbon substrates, an uncommon feature for bacteria of this genus. Furthermore, Pseudomonas sp. MPC6 exhibits a high-copy number of genes encoding for enzymes involved in oxidative and cold-stress response that allows it to cope with high concentrations of heavy metals (As, Cd, Cu) and low temperatures, a finding that was further validated experimentally. We then assessed the growth performance of MPC6 on glycerol using a temperature range from 0 to 45°C, the latter temperature corresponding to the limit at which this Antarctic isolate was no longer able to propagate. On the other hand, the MPC6 genome comprised considerably less virulence and drug resistance factors as compared to pathogenic Pseudomonas strains, thus supporting its safety. Unexpectedly, we found five PHA synthases within the genome of MPC6, one of which clustered separately from the other four. This PHA synthase shared only 40% sequence identity at the amino acid level against the only PHA polymerase described for Pseudomonas (63-1 strain) able to produce copolymers of short- and medium-chain length PHAs. Batch cultures for PHA synthesis in Pseudomonas sp. MPC6 using sugars, decanoate, ethylene glycol, and organic acids as carbon substrates result in biopolymers with different monomer compositions. This indicates that the PHA synthases play a critical role in defining not only the final chemical structure of the biosynthesized PHA, but also the employed biosynthetic pathways. Based on the results obtained, we conclude that Pseudomonas sp. MPC6 can be exploited as a bioremediator and biopolymer factory, as well as a model strain to unveil molecular mechanisms behind adaptation to cold and extreme environments

The Transcription Factor ArcA Modulates Salmonella’s Metabolism in Response to Neutrophil Hypochlorous Acid-Mediated Stress

Indexación: ScopusSalmonella Typhimurium, a bacterial pathogen with high metabolic plasticity, can adapt to different environmental conditions; these traits enhance its virulence by enabling bacterial survival. Neutrophils play important roles in the innate immune response, including the production of microbicidal reactive oxygen species (ROS). In addition, the myeloperoxidase in neutrophils catalyzes the formation of hypochlorous acid (HOCl), a highly toxic molecule that reacts with essential biomolecules, causing oxidative damage including lipid peroxidation and protein carbonylation. The bacterial response regulator ArcA regulates adaptive responses to oxygen levels and influences the survival of Salmonella inside phagocytic cells. Here, we demonstrate by whole transcriptomic analyses that ArcA regulates genes related to various metabolic pathways, enabling bacterial survival during HOCl-stress in vitro. Also, inside neutrophils, ArcA controls the transcription of several metabolic pathways by downregulating the expression of genes related to fatty acid degradation, lysine degradation, and arginine, proline, pyruvate, and propanoate metabolism. ArcA also upregulates genes encoding components of the oxidative pathway. These results underscore the importance of ArcA in ATP generation inside the neutrophil phagosome and its participation in bacterial metabolic adaptations during HOCl stress. © Copyright © 2019 Pardo-Esté, Castro-Severyn, Krüger, Cabezas, Briones, Aguirre, Morales, Baquedano, Sulbaran, Hidalgo, Meneses, Poblete-Castro, Castro-Nallar, Valvano and Saavedra.https://www.frontiersin.org/articles/10.3389/fmicb.2019.02754/ful

Mare stromal endometrial cells differentially modulate inflammation depending on oestrus cycle status: an in vitro study

The modulation of inflammation is pivotal for uterine homeostasis. Here we evaluated the effect of the oestrus cycle on the expression of pro-inflammatory and anti-inflammatory markers in a cellular model of induced fibrosis. Mare endometrial stromal cells isolated from follicular or mid-luteal phase were primed with 10 ng/mL of TGFβ alone or in combination with either IL1β, IL6, or TNFα (10 ng/mL each) or all together for 24 h. Control cells were not primed. Messenger and miRNA expression were analyzed using real-time quantitative PCR (RT-qPCR). Cells in the follicular phase primed with pro-inflammatory cytokines showed higher expression of collagen-related genes (CTGF, COL1A1, COL3A1, and TIMP1) and mesenchymal marker (SLUG, VIM, CDH2, and CDH11) genes; p < 0.05. Cells primed during the mid-luteal overexpressed genes associated with extracellular matrix, processing, and prostaglandin E synthase (MMP2, MMP9, PGR, TIMP2, and PTGES; p < 0.05). There was a notable upregulation of pro-fibrotic miRNAs (miR17, miR21, and miR433) in the follicular phase when the cells were exposed to TGFβ + IL1β, TGFβ + IL6 or TGFβ + IL1β + IL6 + TNFα. Conversely, in cells from the mid-luteal phase, the treatments either did not or diminished the expression of the same miRNAs. On the contrary, the anti-fibrotic miRNAs (miR26a, miR29b, miR29c, miR145, miR378, and mir488) were not upregulated with treatments in the follicular phase. Rather, they were overexpressed in cells from the mid-luteal phase, with the highest regulation observed in TGFβ + IL1β + IL6 + TNFα treatment groups. These miRNAs were also analyzed in the extracellular vesicles secreted by the cells. A similar trend as seen with cellular miRNAs was noted, where anti-fibrotic miRNAs were downregulated in the follicular phase, while notably elevated pro-fibrotic miRNAs were observed in extracellular vesicles originating from the follicular phase. Pro-inflammatory cytokines may amplify the TGFβ signal in the follicular phase resulting in significant upregulation of extracellular matrix-related genes, an imbalance in the metalloproteinases, downregulation of estrogen receptors, and upregulation of pro-fibrotic factors. Conversely, in the luteal phase, there is a protective role mediated primarily through an increase in anti-fibrotic miRNAs, a decrease in SMAD2 phosphorylation, and reduced expression of fibrosis-related genes