| 1. |
- Caspeta-Guadarrama, Luis, 1974, et al.
(författare)
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Altered sterol composition renders yeast thermotolerant
- 2014
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 346:6205, s. 75-78
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Tidskriftsartikel (refereegranskat)abstract
- Ethanol production for use as a biofuel is mainly achieved through simultaneous saccharification and fermentation by yeast. Operating at >= 40 degrees C would be beneficial in terms of increasing efficiency of the process and reducing costs, but yeast does not grow efficiently at those temperatures. We used adaptive laboratory evolution to select yeast strains with improved growth and ethanol production at >= 40 degrees C. Sequencing of the whole genome, genome-wide gene expression, and metabolic-flux analyses revealed a change in sterol composition, from ergosterol to fecosterol, caused by mutations in the C-5 sterol desaturase gene, and increased expression of genes involved in sterol biosynthesis. Additionally, large chromosome III rearrangements and mutations in genes associated with DNA damage and respiration were found, but contributed less to the thermotolerant phenotype.
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| 2. |
- Caspeta-Guadarrama, Luis, 1974, et al.
(författare)
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Economic and environmental impacts of microbial biodiesel
- 2013
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Ingår i: Nature Biotechnology. - : Springer Science and Business Media LLC. - 1087-0156 .- 1546-1696. ; 31:9, s. 789-793
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 3. |
- Caspeta-Guadarrama, Luis, 1974, et al.
(författare)
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Enzymatic hydrolysis at high-solids loadings for the conversion of agave bagasse to fuel ethanol
- 2014
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Ingår i: Applied Energy. - : Elsevier BV. - 1872-9118 .- 0306-2619. ; 113, s. 277-286
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Tidskriftsartikel (refereegranskat)abstract
- Agave bagasse is the lignocellulosic residue accumulated during the production of alcoholic beverages in Mexico and is a potential feedstock for the production of biofuels. A factorial design was used to investigate the effect of temperature, residence time and concentrations of acid and ethanol on ethanosolv pretreatment and enzymatic hydrolysis of agave bagasse. This method and the use of a stirred in-house-made mini-reactor increased the digestibility of agave bagasse from 30% observed with the dilute-acid method to 98%; also allowed reducing the quantity of enzymes used to hydrolyze samples with solid loadings of 30%. w/w and glucose concentrations up to 225. g/L were obtained in the enzymatic hydrolysates. Overall this process allows the recovery of 91% of the total fermentable sugars contained in the agave bagasse (0.51. g/g) and 69% of total lignin as co-product (0.11. g/g). The maximum ethanol yield under optimal conditions using an industrial yeast strain for the fermentation was 0.25. g/g of dry agave bagasse, which is 86% of the maximum theoretical (0.29. g/g). The effect of the glucose concentration and solid loading on the conversion of cellulose to glucose is discussed, in addition to prospective production of about 50. million liters of fuel ethanol using agave bagasse residues from the tequila industry as a potential solution to the disposal problems.
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| 4. |
- Caspeta-Guadarrama, Luis, 1974, et al.
(författare)
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Genome-scale metabolic reconstructions of Pichia stipitis and Pichia pastoris and in-silico evaluation of their potentials
- 2012
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Ingår i: BMC Systems Biology. - : Springer Science and Business Media LLC. - 1752-0509. ; 6:24
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundPichia stipitis and Pichia pastoris have long been investigated due to their native abilities to metabolize every sugar from lignocellulose and to modulate methanol consumption, respectively. The latter has been driving the production of several recombinant proteins. As a result, significant advances in their biochemical knowledge, as well as in genetic engineering and fermentation methods have been generated. The release of their genome sequences has allowed systems level research. ResultsIn this work, genome-scale metabolic models (GEMs) of P. stipitis (iSS884) and P. pastoris (iLC915) were reconstructed. iSS884 includes 1332 reactions, 922 metabolites, and 4 compartments. iLC915 contains 1423 reactions, 899 metabolites, and 7 compartments. Compared with the previous GEMs of P. pastoris, PpaMBEL1254 and iPP668, iLC915 contains more genes and metabolic functions, as well as improved predictive capabilities. Simulations of physiological responses for the growth of both yeasts on selected carbon sources using iSS884 and iLC915 closely reproduced the experimental data. Additionally, the iSS884 model was used to predict ethanol production from xylose at different oxygen uptake rates. Simulations with iLC915 closely reproduced the effect of oxygen uptake rate on physiological states of P. pastoris expressing a recombinant protein. The potential of P. stipitis for the conversion of xylose and glucose into ethanol using reactors in series, and of P. pastoris to produce recombinant proteins using mixtures of methanol and glycerol or sorbitol are also discussed. ConclusionsIn conclusion the first GEM of P. stipitis (iSS884) was reconstructed and validated. The expanded version of the P. pastoris GEM, iLC915, is more complete and has improved capabilities over the existing models. Both GEMs are useful frameworks to explore the versatility of these yeasts and to capitalize on their biotechnological potentials.
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| 5. |
- Caspeta-Guadarrama, Luis, 1974
(författare)
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High cell-density cultivation in batch mode for plasmid DNA production by a metabolically engineered E. coli strain with minimized overflow metabolism
- 2011
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Ingår i: Biochemical Engineering Journal. - 1873-295X .- 1369-703X. ; 56:3, s. 165-171
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Tidskriftsartikel (refereegranskat)abstract
- Progress on plasmid-based (pDNA) vaccines requires simpler and efficient cultivation techniques for their production. A prevalent problem in the cultivation of Escherichia coli (the main host for pDNA vaccines production) is the aerobic production of acetate. In this work, a metabolically engineered Escherichia coli strain with strongly reduced acetate formation was tested for the production of a plasmid vaccine at high cell-densities. The wild type (W3110) and engineered (VH33) strains were cultivated in batch mode using 100g/L of initial glucose concentration. This elevated amount of glucose allowed attaining high cell-densities of strain VH33 without external substrate feeding, simplifying the cultivation process. While W3110 produced 17mg/L of pDNA and 5.3g/L of acetate, VH33 reached 40mg/L of pDNA and only 2g/L of acetate. While the plasmid supercoiling degree progressively decreased in W3110 cultivations, it remained nearly constant for VH33. These results show the successful application of cell engineering concepts for improving DNA vaccine production processes.
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| 6. |
- Caspeta-Guadarrama, Luis, 1974, et al.
(författare)
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Modifying yeast tolerance to inhibitory conditions of ethanol production processes
- 2015
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Ingår i: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media SA. - 2296-4185. ; 3:Nov, s. 184-
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Forskningsöversikt (refereegranskat)abstract
- Saccharomyces cerevisiae strains having a broad range of substrate utilization, rapid substrate consumption, and conversion to ethanol, as well as good tolerance to inhibitory conditions are ideal for cost-competitive ethanol production from lignocellulose. A major drawback to directly design S. cerevisiae tolerance to inhibitory conditions of lignocellulosic ethanol production processes is the lack of knowledge about basic aspects of its cellular signaling network in response to stress. Here, we highlight the inhibitory conditions found in ethanol production processes, the targeted cellular functions, the key contributions of integrated -omics analysis to reveal cellular stress responses according to these inhibitors, and current status on design-based engineering of tolerant and efficient S. cerevisiae strains for ethanol production from lignocellulose.
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| 7. |
- Caspeta-Guadarrama, Luis, 1974
(författare)
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The effect of heating rate on Escherichia coli metabolism, physiological stress, transcriptional response, and production of temperature-induced recombinant protein: a scale-down study
- 2009
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Ingår i: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 102:2, s. 468-
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Tidskriftsartikel (refereegranskat)abstract
- At the laboratory scale, sudden step increases from 30 to 42°C can be readily accomplished when expressing heterologous proteins in heat-inducible systems. However, for large scale-cultures only slow ramp-type increases in temperature are possible due to heat transfer limitations, where the heating rate decreases as the scale increases. In this work, the transcriptional and metabolic responses of a recombinant Escherichia coli strain to temperature-induced synthesis of pre-proinsulin in high cell density cultures were examined at different heating rates. Heating rates of 6, 1.7, 0.8, and 0.4°C/min were tested in a scale-down approach to mimic fermentors of 0.1, 5, 20, and 100 m3, respectively. The highest yield and concentration of recombinant protein was obtained for the slowest heating rate. As the heating rate increased, the yield and maximum recombinant protein concentration decreased, whereas a larger fraction of carbon skeletons was lost as acetate, lactate, and formate. Compared to 30°C, the mRNA levels of selected heat-shock genes at 38 and 42°C, as quantified by qRT-PCR, increased between 2- to over 42-fold when cultures were induced at 6, 1.7, and 0.8°C/min, but no increase was observed at 0.4°C/min. Only small increases (between 1.5- and 4-fold) in the expression of the stress genes spoT and relA were observed at 42°C for cultures induced at 1.7 and 6°C/min, suggesting that cells subjected to slow temperature increases can adapt to stress. mRNA levels of genes from the transcription–translation machinery (tufB, rpoA, and tig) decreased between 40% and 80% at 6, 1.7 and 0.8°C/min, whereas a transient increase occurred for 0.4°C/min at 42°C. mRNA levels of the gene coding for pre-proinsulin showed a similar profile to transcripts of heat-shock genes, reflecting a probable analogous induction mechanism. Altogether, the results obtained indicate that slow heating rates, such as those likely to occur in conventional large-scale fermentors, favored heterologous protein synthesis by the thermo-inducible expression system used in this report. Knowledge of the effect of heating rate on bacterial physiology and product formation is useful for the rational design of scale-down and scale-up strategies and optimum recombinant protein induction schemes. Biotechnol. Bioeng. 2009;102: 468–482. © 2008 Wiley Periodicals, Inc.
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| 8. |
- Caspeta-Guadarrama, Luis, 1974, et al.
(författare)
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The role of biofuels in the future energy supply
- 2013
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Ingår i: Energy and Environmental Sciences. - 1754-5692 .- 1754-5706. ; 6:4, s. 1077-1082
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Forskningsöversikt (refereegranskat)abstract
- In recent years several different arguments have been raised against the use of biofuels and their role in our future energy supply. These arguments can be divided into issues related to costs, food versus fuel, and lack of sustainability. Here we address these three points and argue that biofuels represent an essential contribution to our future energy supply and more importantly will contribute to a reduction in carbon dioxide emissions.
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| 9. |
- Caspeta-Guadarrama, Luis, 1974, et al.
(författare)
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The yeastGemMap: A process diagram to assist yeast systems-metabolic studies
- 2021
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 118:12, s. 4800-4814
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Tidskriftsartikel (refereegranskat)abstract
- Visualization is a key aspect of the analysis of omics data. Although many tools can generate pathway maps for visualization of yeast metabolism, they fail in reconstructing genome-scale metabolic diagrams of compartmentalized metabolism. Here we report on the yeastGemMap, a process diagram of whole yeast metabolism created to assist data analysis in systems-metabolic studies. The map was manually reconstructed with reactions from a compartmentalized genome-scale metabolic model, based on biochemical process diagrams typically found in educational and specialized literature. The yeastGemMap consists of 3815 reactions representing 1150 genes, 2742 metabolites, and 14 compartments. Computational functions for adapting the graphical representation of the map are also reported. These functions modify the visual representation of the map to assist in three systems-metabolic tasks: illustrating reaction networks, interpreting metabolic flux data, and visualizing omics data. The versatility of the yeastGemMap and algorithms to assist visualization of systems-metabolic data was demonstrated in various tasks, including for single lethal reaction evaluation, flux balance analysis, and transcriptomic data analysis. For instance, visual interpretation of metabolic transcriptomes of thermally evolved and parental yeast strains allowed to demonstrate that evolved strains activate a preadaptation response at 30 degrees C, which enabled thermotolerance. A quick interpretation of systems-metabolic data is promoted with yeastGemMap visualizations.
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| 10. |
- Caspeta-Guadarrama, Luis, 1974, et al.
(författare)
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Thermotolerant Yeast Strains Adapted by Laboratory Evolution Show Trade-Off at Ancestral Temperatures and Preadaptation to Other Stresses
- 2015
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Ingår i: mBio. - 2150-7511 .- 2161-2129. ; 6:4
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Tidskriftsartikel (refereegranskat)abstract
- A major challenge for the production of ethanol from biomass-derived feedstocks is to develop yeasts that can sustain growth under the variety of inhibitory conditions present in the production process, e.g., high osmolality, high ethanol titers, and/or elevated temperatures (>40 degrees C). Using adaptive laboratory evolution, we previously isolated seven Saccharomyces cerevisiae strains with improved growth at 40 degrees C. Here, we show that genetic adaptations to high temperature caused a growth trade-off at ancestral temperatures, reduced cellular functions, and improved tolerance of other stresses. Thermotolerant yeast strains showed horizontal displacement of their thermal reaction norms to higher temperatures. Hence, their optimal and maximum growth temperatures increased by about 3 degrees C, whereas they showed a growth trade-off at temperatures below 34 degrees C. Computational analysis of the physical properties of proteins showed that the lethal temperature for yeast is around 49 degrees C, as a large fraction of the yeast proteins denature above this temperature. Our analysis also indicated that the number of functions involved in controlling the growth rate decreased in the thermotolerant strains compared with the number in the ancestral strain. The latter is an advantageous attribute for acquiring thermotolerance and correlates with the reduction of yeast functions associated with loss of respiration capacity. This trait caused glycerol overproduction that was associated with the growth trade-off at ancestral temperatures. In combination with altered sterol composition of cellular membranes, glycerol overproduction was also associated with yeast osmotolerance and improved tolerance of high concentrations of glucose and ethanol. Our study shows that thermal adaptation of yeast is suitable for improving yeast resistance to inhibitory conditions found in industrial ethanol production processes. IMPORTANCE Yeast thermotolerance can significantly reduce the production costs of biomass conversion to ethanol. However, little information is available about the underlying genetic changes and physiological functions required for yeast thermotolerance. We recently revealed the genetic changes of thermotolerance in thermotolerant yeast strains (TTSs) generated through adaptive laboratory evolution. Here, we examined these TTSs' physiology and computed their proteome stability over the entire thermal niche, as well as their preadaptation to other stresses. Using this approach, we showed that TTSs exhibited evolutionary trade-offs in the ancestral thermal niche, as well as reduced numbers of growth functions and preadaptation to other stresses found in ethanol production processes. This information will be useful for rational engineering of yeast thermotolerance for the production of biofuels and chemicals.
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| 11. |
- Caspeta-Guadarrama, Luis, 1974, et al.
(författare)
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Thermotolerant yeasts selected by adaptive evolution express heat stress response at 30 degrees C
- 2016
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Exposure to long-term environmental changes across >100s of generations results in adapted phenotypes, but little is known about how metabolic and transcriptional responses are optimized in these processes. Here, we show that thermotolerant yeast strains selected by adaptive laboratory evolution to grow at increased temperature, activated a constitutive heat stress response when grown at the optimal ancestral temperature, and that this is associated with a reduced growth rate. This preventive response was perfected by additional transcriptional changes activated when the cultivation temperature is increased. Remarkably, the sum of global transcriptional changes activated in the thermotolerant strains when transferred from the optimal to the high temperature, corresponded, in magnitude and direction, to the global changes observed in the ancestral strain exposed to the same transition. This demonstrates robustness of the yeast transcriptional program when exposed to heat, and that the thermotolerant strains streamlined their path to rapidly and optimally reach post-stress transcriptional and metabolic levels. Thus, long-term adaptation to heat improved yeasts ability to rapidly adapt to increased temperatures, but this also causes a trade-off in the growth rate at the optimal ancestral temperature.
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| 12. |
- Caspeta-Guadarrama, Luis, 1974, et al.
(författare)
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Toward systems metabolic engineering of Aspergillus and Pichia species for the production of chemicals and biofuels
- 2013
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Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 8:5, s. 534-544
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Forskningsöversikt (refereegranskat)abstract
- Recently genome sequence data have become available for Aspergillus and Pichia species of industrial interest. This has stimulated the use of systems biology approaches for large-scale analysis of the molecular and metabolic responses of Aspergillus and Pichia under defined conditions, which has resulted in much new biological information. Case-specific contextualization of this information has been performed using comparative and functional genomic tools. Genomics data are also the basis for constructing genome-scale metabolic models, and these models have helped in the contextualization of knowledge on the fundamental biology of Aspergillus and Pichia species. Furthermore, with the availability of these models, the engineering of Aspergillus and Pichia is moving from traditional approaches, such as random mutagenesis, to a systems metabolic engineering approach. Here we review the recent trends in systems biology of Aspergillus and Pichia species, highlighting the relevance of these developments for systems metabolic engineering of these organisms for the production of hydrolytic enzymes, biofuels and chemicals from biomass.
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| 13. |
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