Examinando por Autor "Poblete-Castro, I."
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Ítem A novel programmable lysozyme-based lysis system in Pseudomonas putida for biopolymer production(Nature Publishing Group, 2017-06) Borrero de Acuña, J.M.; Hidalgo-Dumont, C.; Pacheco, N.; Cabrera, A.; Poblete-Castro, I.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.Ítem Batch cultivation model for biopolymer production(Assoc. of Chemists and Chemical Engineers of Croatia, 2017) Torres-Cerna, C.E.; Alanis, A.Y.; Poblete-Castro, I.; Hernandez-Vargas, E.A.This paper presents a mathematical model to evaluate the kinetics of two different Pseudomonas putida strains, wild and mutant-type for the microbial production of polyhydroxyalkanoates (PHAs). Model parameters were estimated to represent adequately experimental data from the batch reactor using the differential evolution algorithm. Based on the mathematical model with the best-fit parameter values, simulations suggested that the high production of PHA by the mutant strain can be attributed not only to the higher production of PHA but also to a reduction in the consumption rate of the substrates of approximately 66 %. Remarkably, the cell growth rate value is higher for the wild type than the mutant type, suggesting that the PHA increase is not only to an increase in the production rate but also to the metabolism of the cells. This mathematical model advances comprehension of the PHA production capacity by P. putida paving the road towards environmentally friendly plastics.Ítem Biochemistry, genetics and biotechnology of glycerol utilization in Pseudomonas species(John Wiley and Sons Ltd, 2020) Poblete-Castro, I.; Wittmann, C.; Nikel, P.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.Ítem Cascaded valorization of seaweed using microbial cell factories(Elsevier Ltd, 2020-10) Poblete-Castro, I.; Hoffmann, S.-L.; Becker, J.; Wittmann, C.Sustainable production from seaweed has grown into an area of intense research and development. Meanwhile, more than 30 million tonnes of seaweed are produced, of which 70% are used as food and 30% have other applications such as feed, fertilizer, chemicals, and energy. Towards biorefining seaweed in an environmentally friendly and economically viable manner, we need efficient approaches that convert its biomass and residuals into added value products. Smart cell factories and fermentation strategies which can be integrated into future seaweed biorefineries are at the heart of the development and therefore receive increasing attention. Here, we review advances in the field including novel fermentation routes from seaweed to chemicals, materials, pharmaceuticals, fuels and energy, and discuss challenges and opportunities. © 2020 The AuthorsÍtem Datasets for transcriptomics, q-proteomics and phenotype microarrays of polyphosphate metabolism mutants from Escherichia coli(ELSEVIER SCIENCE, 2017-06) Varas, M.; Valdivieso, C.; Mauriaca, C.; Ortíz-Severín, J.; Paradela, A.; Poblete-Castro, I.; Cabrera, R.; Chávez, F.P.Here, 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.Ítem 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(Elsevier, 2019-02) Borrero-de Acuña, J.M.; Aravena-Carrasco, C.; Gutierrez-Urrutia, I.; Duchens, D.; Poblete-Castro, I.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.Ítem Fed-Batch mcl- Polyhydroxyalkanoates Production in Pseudomonas putida KT2440 and ΔphaZ Mutant on Biodiesel-Derived Crude Glycerol(Frontiers Media S.A., 2021-03) Borrero-de Acuña, J.M.; Rohde, M.; Saldias, C.; Poblete-Castro, I.Crude 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.Ítem Limited life cycle and cost assessment for the bioconversion of lignin-derived aromatics into adipic acid(John Wiley and Sons Inc., 2020-05) van Duuren, J.B.J.H.; de Wild, P.J.; Starck, S.; Bradtmöller, C.; Selzer, M.; Mehlmann, K.; Schneider, R.; Kohlstedt, M.; Poblete-Castro, I.; Stolzenberger, J.; Barton, N.; Fritz, M.; Scholl, S.; Venus, J.; Wittmann, C.Lignin is an abundant and heterogeneous waste byproduct of the cellulosic industry, which has the potential of being transformed into valuable biochemicals via microbial fermentation. In this study, we applied a fast-pyrolysis process using softwood lignin resulting in a two-phase bio-oil containing monomeric and oligomeric aromatics without syringol. We demonstrated that an additional hydrodeoxygenation step within the process leads to an enhanced thermochemical conversion of guaiacol into catechol and phenol. After steam bath distillation, Pseudomonas putida KT2440-BN6 achieved a percent yield of cis, cis-muconic acid of up to 95 mol% from catechol derived from the aqueous phase. We next established a downstream process for purifying cis, cis-muconic acid (39.9 g/L) produced in a 42.5 L fermenter using glucose and benzoate as carbon substrates. On the basis of the obtained values for each unit operation of the empirical processes, we next performed a limited life cycle and cost analysis of an integrated biotechnological and chemical process for producing adipic acid and then compared it with the conventional petrochemical route. The simulated scenarios estimate that by attaining a mixture of catechol, phenol, cresol, and guaiacol (1:0.34:0.18:0, mol ratio), a titer of 62.5 (g/L) cis, cis-muconic acid in the bioreactor, and a controlled cooling of pyrolysis gases to concentrate monomeric aromatics in the aqueous phase, the bio-based route results in a reduction of CO2-eq emission by 58% and energy demand by 23% with a contribution margin for the aqueous phase of up to 88.05 euro/ton. We conclude that the bio-based production of adipic acid from softwood lignins brings environmental benefits over the petrochemical procedure and is cost-effective at an industrial scale. Further research is essential to achieve the proposed cis, cis-muconic acid yield from true lignin-derived aromatics using whole-cell biocatalysts. © 2020 Wiley Periodicals, Inc.