| 1. |
- Arnling Bååth, Jenny, 1987, et al.
(author)
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Structure-function analysis of two closely related cutinases from Thermobifida cellulosilytica
- 2022
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In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 119:2, s. 470-481
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Journal article (peer-reviewed)abstract
- Cutinases can play a significant role in a biotechnology-based circular economy. However, relatively little is known about the structure–function relationship of these enzymes, knowledge that is vital to advance optimized, engineered enzyme candidates. Here, two almost identical cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) with only 18 amino acids difference were used for a rigorous biochemical characterization of their ability to hydrolyze poly(ethylene terephthalate) (PET), PET-model substrates, and cutin-model substrates. Kinetic parameters were compared with detailed in silico docking studies of enzyme-ligand interactions. The two enzymes interacted with, and hydrolyzed PET differently, with Thc_Cut1 generating smaller PET-degradation products. Thc_Cut1 also showed higher catalytic efficiency on long-chain aliphatic substrates, an effect likely caused by small changes in the binding architecture. Thc_Cut2, in contrast, showed improved binding and catalytic efficiency when approaching the glass transition temperature of PET, an effect likely caused by longer amino acid residues in one area at the enzyme's surface. Finally, the position of the single residue Q93 close to the active site, rotated out in Thc_Cut2, influenced the ligand position of a trimeric PET-model substrate. In conclusion, we illustrate that even minor sequence differences in cutinases can affect their substrate binding, substrate specificity, and catalytic efficiency drastically.
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| 2. |
- Brechmann, Nils A., et al.
(author)
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Antibody capture process based on magnetic beads from very high cell density suspension
- 2021
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In: Biotechnology and Bioengineering. - : John Wiley and Sons Inc. - 0006-3592 .- 1097-0290. ; 118:9, s. 3499-3510
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Journal article (peer-reviewed)abstract
- Cell clarification represents a major challenge for the intensification through very high cell density in the production of biopharmaceuticals such as monoclonal antibodies (mAbs). The present report proposes a solution to this challenge in a streamlined process where cell clarification and mAb capture are performed in a single step using magnetic beads coupled with protein A. Capture of mAb from non-clarified CHO cell suspension showed promising results; however, it has not been demonstrated that it can handle the challenge of very high cell density as observed in intensified fed-batch cultures. The performances of magnetic bead-based mAb capture on non-clarified cell suspension from intensified fed-batch culture were studied. Capture from a culture at density larger than 100 × 106 cells/ml provided an adsorption efficiency of 99% and an overall yield of 93% with a logarithmic host cell protein (HCP) clearance of ≈2–3 and a resulting HCP concentration ≤≈5 ppm. These results show that direct capture from very high cell density cell suspension is possible without prior processing. This technology, which brings significant benefits in terms of operational cost reduction and performance improvements such as low HCP, can be a powerful tool alleviating the challenge of process intensification.
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| 3. |
- Cabaneros Lopez, Pau, et al.
(author)
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Transforming data to information : A parallel hybrid model for real-time state estimation in lignocellulosic ethanol fermentation
- 2021
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In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 118:2, s. 579-591
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Journal article (peer-reviewed)abstract
- Operating lignocellulosic fermentation processes to produce fuels and chemicals is challenging due to the inherent complexity and variability of the fermentation media. Real-time monitoring is necessary to compensate for these challenges, but the traditional process monitoring methods fail to deliver actionable information that can be used to implement advanced control strategies. In this study, a hybrid-modeling approach is presented to monitor cellulose-to-ethanol (EtOH) fermentations in real-time. The hybrid approach uses a continuous-discrete extended Kalman filter to reconciliate the predictions of a data-driven model and a kinetic model and to estimate the concentration of glucose (Glu), xylose (Xyl), and EtOH. The data-driven model is based on partial least squares (PLS) regression and predicts in real-time the concentration of Glu, Xyl, and EtOH from spectra collected with attenuated total reflectance mid-infrared spectroscopy. The estimations made by the hybrid approach, the data-driven models and the internal model were compared in two validation experiments showing that the hybrid model significantly outperformed the PLS and improved the predictions of the internal model. Furthermore, the hybrid model delivered consistent estimates even when disturbances in the measurements occurred, demonstrating the robustness of the method. The consistency of the proposed hybrid model opens the doors towards the implementation of advanced feedback control schemes.
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| 4. |
- Caspeta-Guadarrama, Luis, 1974, et al.
(author)
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The yeastGemMap: A process diagram to assist yeast systems-metabolic studies
- 2021
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In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 118:12, s. 4800-4814
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Journal article (peer-reviewed)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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| 5. |
- Chen, Yu, 1990, et al.
(author)
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Genome-scale modeling for Bacillus coagulans to understand the metabolic characteristics
- 2020
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In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 117:11, s. 3545-3558
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Journal article (peer-reviewed)abstract
- Lactic acid is widely used in many industries, especially in the production of poly-lactic acid. Bacillus coagulans is a promising lactic acid producer in industrial fermentation due to its thermophilic property. In this study, we developed the first genome-scale metabolic model (GEM) of B. coagulans iBag597, together with an enzyme-constrained model ec-iBag597. We measured strain-specific biomass composition and integrated the data into a biomass equation. Then, we validated iBag597 against experimental data generated in this study, including amino acid requirements and carbon source utilization, showing that simulations were generally consistent with the experimental results. Subsequently, we carried out chemostats to investigate the effects of specific growth rate and culture pH on metabolism of B. coagulans. Meanwhile, we used iBag597 to estimate the intracellular metabolic fluxes for those conditions. The results showed that B. coagulans was capable of generating ATP via multiple pathways, and switched among them in response to various conditions. With ec-iBag597, we estimated the protein cost and protein efficiency for each ATP-producing pathway to investigate the switches. Our models pave the way for systems biology of B. coagulans, and our findings suggest that maintaining a proper growth rate and selecting an optimal pH are beneficial for lactate fermentation.
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| 6. |
- Ferreira, Sofia, et al.
(author)
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Metabolic engineering strategies for butanol production in Escherichia coli
- 2020
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In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 117:8, s. 2571-2587
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Research review (peer-reviewed)abstract
- The global market of butanol is increasing due to its growing applications as solvent, flavoring agent, and chemical precursor of several other compounds. Recently, the superior properties of n-butanol as a biofuel over ethanol have stimulated even more interest. (Bio)butanol is natively produced together with ethanol and acetone by Clostridium species through acetone-butanol-ethanol fermentation, at noncompetitive, low titers compared to petrochemical production. Different butanol production pathways have been expressed in Escherichia coli, a more accessible host compared to Clostridium species, to improve butanol titers and rates. The bioproduction of butanol is here reviewed from a historical and theoretical perspective. All tested rational metabolic engineering strategies in E. coli to increase butanol titers are reviewed: manipulation of central carbon metabolism, elimination of competing pathways, cofactor balancing, development of new pathways, expression of homologous enzymes, consumption of different substrates, and molecular biology strategies. The progress in the field of metabolic modeling and pathway generation algorithms and their potential application to butanol production are also summarized here. The main goals are to gather all the strategies, evaluate the respective progress obtained, identify, and exploit the outstanding challenges.
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| 7. |
- Han, Yilin, et al.
(author)
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Molecular genetic analysis of neural stem cells after space flight and simulated microgravity on earth
- 2021
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In: Biotechnology and Bioengineering. - : John Wiley & Sons. - 0006-3592 .- 1097-0290. ; 118:10, s. 3832-3846
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Journal article (peer-reviewed)abstract
- Understanding how stem cells adapt to space flight conditions is fundamental for human space missions and extraterrestrial settlement. We analyzed gene expression in boundary cap neural crest stem cells (BCs), which are attractive for regenerative medicine by their ability to promote proliferation and survival of cocultured and co-implanted cells. BCs were launched to space (space exposed cells) (SEC), onboard sounding rocket MASER 14 as free-floating neurospheres or in a bioprinted scaffold. For comparison, BCs were placed in a random positioning machine (RPM) to simulate microgravity on earth (RPM cells) or were cultured under control conditions in the laboratory. Using next-generation RNA sequencing and data post-processing, we discovered that SEC upregulated genes related to proliferation and survival, whereas RPM cells upregulated genes associated with differentiation and inflammation. Thus, (i) space flight provides unique conditions with distinctly different effects on the properties of BC compared to earth controls, and (ii) the space flight exposure induces postflight properties that reinforce the utility of BC for regenerative medicine and tissue engineering.
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| 8. |
- He, Niling, et al.
(author)
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Cyclic l-lactide synthesis from lignocellulose biomass by biorefining with complete inhibitor removal and highly simultaneous sugars assimilation
- 2022
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In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 119:7, s. 1903-1915
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Journal article (peer-reviewed)abstract
- Cyclic chiral lactide is the monomer chemical for polymerization of high molecular weight polylactic acid (PLA). The synthesis of cyclic l-lactide starts from poly-condensation of l-lactic acid to a low molecular weight prepolymer and then depolymerized to cyclic l-lactide. Lignocellulose biomass is the most promising carbohydrate feedstock for lactic acid production, but the synthesis of cyclic l-lactide from l-lactic acid produced from lignocellulose has so far not been successful. The major barriers are the impurities of residual sugars and inhibitors in the crude cellulosic l-lactic acid product. Here we show a successful cyclic l-lactide synthesis from cellulosic l-lactic acid by lignocellulose biorefining with complete inhibitor removal and coordinated sugars assimilation. The removal of inhibitors from lignocellulose pretreatment was accomplished by biodetoxification using a unique fungus Amorphotheca resinae ZN1. The nonglucose sugars were completely and simultaneously assimilated at the same rate with glucose by the engineered l-lactic acid bacterium Pediococcus acidilactici. The l-lactic acid production from wheat straw was comparable to that from corn starch with high optical pure (99.6%), high l-lactic acid titer (129.4 g/L), minor residual total sugars (~2.2 g/L), and inhibitors free. The cyclic l-lactide was successfully synthesized from the regularly purified l-lactic acid and verified by detailed characterizations. This study paves the technical foundation of carbon-neutral production of biodegradable PLA from lignocellulose biomass.
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| 9. |
- Karimi, Mohsen, 1983, et al.
(author)
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Towards enhancement of gas–liquid mass transfer in bioelectrochemical systems: Validation of a robust CFD model
- 2021
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In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 118:10, s. 3953-3961
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Journal article (peer-reviewed)abstract
- Mass transfer has been identified as a major bottleneck in gas fermentation and microbial conversion of carbon dioxide to chemicals. We present a pragmatic and validated Computational Fluid Dynamics (CFD) model for mass transfer in bioelectrochemical systems. Experiments were conducted to measure mixing times and mass transfer in a Duran bottle and an H-cell. An Eulerian–Eulerian framework with a simplified model for the bubble size distribution (BSD) was developed that utilized only one additional equation for the bubble number density while including the breakup and coalescence. Validations of the CFD model for mixing times showed that the predictions were within the confidence intervals of the measurements, verifying the model's capability in simulating the hydrodynamics. Further validations were performed using constant and varying bubble diameters for the mass transfer. The results showed the benefits of a simplified BSD model, as it yielded improvements of seven and four times in accuracy when assessed against the experimental data for the Duran bottle and H-cell, respectively. Modeling of the H-cell predicted that a lower stirring rate improves mass transfer compared with higher stirring rates, which is of great importance when designing microbial cultivation processes. The model offers a feasible framework for advanced modeling of gas fermentation and microbial electrosynthesis.
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| 10. |
- Kim, Young-Seok, et al.
(author)
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Built-in RNA-mediated chaperone (chaperna) for antigen folding tailored to immunized hosts
- 2020
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In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 117:7, s. 1990-2007
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Journal article (peer-reviewed)abstract
- High-quality antibody (Ab) production depends on the availability of immunologically relevant antigens. We present a potentially universal platform for generating soluble antigens from bacterial hosts, tailored to immunized animals for Ab production. A novel RNA-dependent chaperone, in which the target antigen is genetically fused with an RNA-interacting domain (RID) docking tag derived from the immunized host, promotes the solubility and robust folding of the target antigen. We selected the N-terminal tRNA-binding domain of lysyl-tRNA synthetase (LysRS) as the RID for fusion with viral proteins and demonstrated the expression of the RID fusion proteins in their soluble and native conformations; immunization predominantly elicited Ab responses to the target antigen, whereas the self RID tag remained nonimmunogenic. Differential immunogenicity of the fusion proteins greatly enriched and simplified the screening of hybridoma clones of monoclonal antibodies (mAbs), enabling specific and sensitive serodiagnosis of MERS-CoV infection. Moreover, mAbs against the consensus influenza hemagglutinin stalk domain enabled a novel assay for trivalent seasonal influenza vaccines. The Fc-mediated effector function was demonstrated, which could be harnessed for the design of next-generation universal influenza vaccines. The nonimmunogenic built-in antigen folding module tailored to a repertoire of immunized animal hosts will drive immunochemical diagnostics, therapeutics, and designer vaccines.
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