Effective application of immobilized second generation industrial Saccharomyces cerevisiae strain on consolidated bioprocessing.

Integrated bioprocessing strategies can facilitate ethanol production from both cellulose and hemicellulose fractions of lignocellulosic biomass. Consolidated bioprocessing (CBP) is an approach that combines enzyme production, biomass hydrolysis and sugar fermentation in a single step. However, technologies that propose the use of microorganisms together with solid biomass present the difficulty of the recovery and reuse of the biocatalyst, which can be overcome by cell immobilization. In this regard, this work applied immobilized cells of AC14 yeast, a recombinant yeast that secretes 7 hydrolytic enzymes, in the CBP process in a successful proof-of-concept for the enzyme access to the substrate polymers. The most appropriate cell load for CBP under the conditions studied with immobilized cells was selected among three optical densities (OD) 10, 55 and 100. These experiments were performed with free cells to ensure that the results were not biased by mass limitations effects. OD 10 achieved 100% of the sugar consumption and the higher specific production of enzymes, being selected for further studies. Diffusional effects were observed with immobilized cells under static conditions. However, mass transfer limitations were mitigated under agitation, with an 18.5% increase in substrate consumption rate (from 2.7 to 3.5 g/L/h), reaching the same substrate uptake rates as free cells. In addition, immobilized cells achieved 100% hydrolysis and consumption of all substrates offered within only 12 h. Overall, this is the first report of a successful application of immobilized yeast cells in CBP processes for bioethanol production, a promising technology that can be extended to other biorefinery bioproducts.

A comprehensive method for modeling and simulating ion exchange chromatography of complex mixtures.

In recent years, many biological-based products have been developed, representing a significant fraction of income in the pharmaceutical market. Ion exchange chromatography is an important downstream step for the purification of target recombinant proteins present in clarified cell extracts, together with many other unknown impurities. This work develops a robust approach to model and simulate the purification of untagged heterologous proteins, so that the improved conditions to carry out an ion exchange chromatography are identified in a rational basis prior to the real purification run itself. Purification of the pneumococcal surface protein A (PspA4Pro) was used as a case study. This protein is produced by recombinant Escherichia coli and is a candidate for the manufacture of improved pneumococcal vaccines. The developed method combined experimental and computational procedures. Different anion exchange operating conditions were mapped in order to gather a broad range of representative experimental data. The equilibrium dispersive and the steric mass action equations were used to model and simulate the process. A training strategy to fit the model and separately describe the elution profiles of PspA4Pro and other proteins of the cell extract was applied. Based on the simulation results, a reduced ionic strength was applied for PspA4Pro elution, leading to increases of 14.9% and 11.5% for PspA4Pro recovery and purity, respectively, compared to the original elution profile. These results showed the potential of this method, which could be further applied to improve the performance of ion exchange chromatography in the purification of other target proteins under real process conditions.

Cell Immobilization Using Alginate-Based Beads as a Protective Technique against Stressful Conditions of Hydrolysates for 2G Ethanol Production.

The development of biorefineries brings the necessity of an efficient consumption of all sugars released from biomasses, including xylose. In addition, the presence of inhibitors in biomass hydrolysates is one of the main challenges in bioprocess feasibility. In this study, the application of Ca-alginate hybrid gels in the immobilization of xylose-consuming recombinant yeast was explored with the aim of improving the tolerance of inhibitors. The recombinant yeast Saccharomyces cerevisiae GSE16-T18SI.1 (T18) was immobilized in Ca-alginate and Ca-alginate-chitosan hybrid beads, and its performance on xylose fermentation was evaluated in terms of tolerance to different acetic acid concentrations (0-12 g/L) and repeated batches of crude sugarcane bagasse hemicellulose hydrolysate. The use of the hybrid gel improved yeast performance in the presence of 12 g/L of acetic acid, achieving 1.13 g/L/h of productivity and reaching 75% of the theoretical ethanol yield, with an improvement of 32% in the xylose consumption rate (1:1 Vbeads/Vmedium, 35 °C, 150 rpm and pH 5.2). The use of hybrid alginate-chitosan gel also led to better yeast performance at crude hydrolysate, yielding one more batch than the pure-alginate beads. These results demonstrate the potential of a hybrid gel as an approach that could increase 2G ethanol productivity and allow cell recycling for a longer period.

ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification.

Several studies have searched for new antigens to produce pneumococcal vaccines that are more effective and could provide broader coverage, given the great number of serotypes causing pneumococcal diseases. One of the promising subunit vaccine candidates is untagged recombinant pneumococcal surface protein A (PspA4Pro), obtainable in high quantities using recombinant Escherichia coli as a microbial factory. However, lipopolysaccharides (LPS) present in E. coli cell extracts must be removed, in order to obtain the target protein at the required purity, which makes the downstream process more complex and expensive. Endotoxin-free E. coli strains, which synthesize a nontoxic mutant LPS, may offer a cost-effective alternative way to produce recombinant proteins for application as therapeutics. This paper presents an investigation of PspA4Pro production employing the endotoxin-free recombinant strain ClearColi® BL21(DE3) with different media (defined, auto-induction, and other complex media), temperatures (27, 32, and 37 °C), and inducers. In comparison to conventional E. coli cells in a defined medium, ClearColi presented similar PspA4Pro yields, with lower productivities. Complex medium formulations supplemented with salts favored PspA4Pro yields, titers, and ClearColi growth rates. Induction with isopropyl-ß-D-thiogalactopyranoside (0.5 mM) and lactose (2.5 g/L) together in a defined medium at 32 °C, which appeared to be a promising cultivation strategy, was reproduced in 5 L bioreactor culture, leading to a yield of 146.0 mg PspA4Pro/g dry cell weight. After purification, the cell extract generated from ClearColi led to 98% purity PspA4Pro, which maintained secondary structure and biological function. ClearColi is a potential host for industrial recombinant protein production. KEY POINTS: • ClearColi can produce as much PspA4Pro as conventional E. coli BL21(DE3) cells. • 10.5 g PspA4Pro produced in ClearColi bioreactor culture using a defined medium. • Functional PspA4Pro (98% of purity) was obtained in ClearColi bioreactor culture. Graphical abstract.

A comprehensive method for modeling and simulating ion exchange chromatography of complex mixtures

In recent years, many biological-based products have been developed, representing a significant fraction of income in the pharmaceutical market. Ion exchange chromatography is an important downstream step for the purification of target recombinant proteins present in clarified cell extracts, together with many other unknown impurities. This work develops a robust approach to model and simulate the purification of untagged heterologous proteins, so that the improved conditions to carry out an ion exchange chromatography are identified in a rational basis prior to the real purification run itself. Purification of the pneumococcal surface protein A (PspA4Pro) was used as a case study. This protein is produced by recombinant Escherichia coli and is a candidate for the manufacture of improved pneumococcal vaccines. The developed method combined experimental and computational procedures. Different anion exchange operating conditions were mapped in order to gather a broad range of representative experimental data. The equilibrium dispersive and the steric mass action equations were used to model and simulate the process. A training strategy to fit the model and separately describe the elution profiles of PspA4Pro and other proteins of the cell extract was applied. Based on the simulation results, a reduced ionic strength was applied for PspA4Pro elution, leading to increases of 14.9% and 11.5% for PspA4Pro recovery and purity, respectively, compared to the original elution profile. These results showed the potential of this method, which could be further applied to improve the performance of ion exchange chromatography in the purification of other target proteins under real process conditions.

ClearColi as a platform for untagged pneumococcal surface protein a production: cultivation strategy, bioreactor culture, and purification

Several studies have searched for new antigens to produce pneumococcal vaccines that are more effective and could provide broader coverage, given the great number of serotypes causing pneumococcal diseases. One of the promising subunit vaccine candidates is untagged recombinant pneumococcal surface protein A (PspA4Pro), obtainable in high quantities using recombinant Escherichia coli as a microbial factory. However, lipopolysaccharides (LPS) present in E. coli cell extracts must be removed, in order to obtain the target protein at the required purity, which makes the downstream process more complex and expensive. Endotoxin-free E. coli strains, which synthesize a nontoxic mutant LPS, may offer a cost-effective alternative way to produce recombinant proteins for application as therapeutics. This paper presents an investigation of PspA4Pro production employing the endotoxin-free recombinant strain ClearColi® BL21(DE3) with different media (defined, auto-induction, and other complex media), temperatures (27, 32, and 37 °C), and inducers. In comparison to conventional E. coli cells in a defined medium, ClearColi presented similar PspA4Pro yields, with lower productivities. Complex medium formulations supplemented with salts favored PspA4Pro yields, titers, and ClearColi growth rates. Induction with isopropyl-β-D-thiogalactopyranoside (0.5 mM) and lactose (2.5 g/L) together in a defined medium at 32 °C, which appeared to be a promising cultivation strategy, was reproduced in 5 L bioreactor culture, leading to a yield of 146.0 mg PspA4Pro/g dry cell weight. After purification, the cell extract generated from ClearColi led to 98% purity PspA4Pro, which maintained secondary structure and biological function. ClearColi is a potential host for industrial recombinant protein production.

Synergy of Aspergillus niger and Components in Biofertilizer Composites Increases the Availability of Nutrients to Plants.

Intensive fertilization has been required to provide nutrients for plant growth under the current agricultural practices being applied to meet the global food demands. Micronutrients such as zinc, manganese, and copper are required in small quantities when compared to macronutrients (such as nitrogen, phosphorus and potassium), but they are essential for the plant growth cycle and consequently for increasing productivity. Mineral oxides such as ZnO, MnO, and CuO are used in agriculture as micronutrient sources, but their low solubility limits practical applications in plant nutrition. Similarly, elemental sulfur (S0) can provide a high-concentration source of sulfate, but its availability is limited by the ability of the soil to promote S0 oxidation. We propose here the integration of these nutrients in a composite based on a biodegradable starch matrix containing mineral oxides and S0 in a dispersion that allowed encapsulation of the acidifying agent Aspergillus niger, a native soil fungus. This strategy effectively improved the final nutrient solubility, with the composite starch/S0/oxidemixture multi-nutrient fertilizer showing remarkable results for solubilization of the oxides, hence confirming a synergic effect of S0 oxidation and microbial solubilization. This composite exhibited an extended shelf life and soil-plant experiments with Italian ryegrass (Lolium multiflorum Lam.) confirmed high efficiencies for dry matter production, nutrient uptake, and recovery. These findings can contribute to the development of environmentally friendly fertilizers towards a more sustainable agriculture and could open up new applications for formulations containing poorly soluble oxide sources.

High stabilization and hyperactivation of a Recombinant ß-Xylosidase through Immobilization Strategies.

Attainment of a stable and highly active ß-xylosidase is of major importance for the efficient and cost-competitive hydrolysis of hemicellulose xylan, as well as for its industrial conversion into biofuels and biochemicals. Here, a recombinant ß-xylosidase of the glycoside hydrolase family (GH43) from Bacillus subtilis was produced in Escherichia coli culture, purified, and subsequently immobilized on agarose and chitosan. Glutaraldehyde and glyoxyl groups were evaluated as activating agents to select the most efficient derivative. Multi-point immobilization on agarose led to an extraordinary thermal stability (half-lives 3604 and 164-fold higher than the free enzyme, at 50° and 35 °C, respectively). Even for chitosan activated with glutaraldehyde, a low-cost support, thermal stability of the immobilized enzyme was 326 and 12-fold higher than the free enzyme at 50° and 35°C, respectively. Immobilized enzymes showed no release of any subunit for the agarose-glyoxyl derivative, and only a few ones for the support activated with glutaraldehyde. Most remarkably, the enzyme kinetic behavior after immobilization increased up to 4-fold in relation to the free one. ß-xylosidase, a tetrameric enzyme with four identical subunits, exists in equilibrium between the monomeric and oligomeric forms in solution. Depending on the pH of immobilization, the enzyme oligomerization can be favored, thus explaining the hyperactivation phenomenon. Both glyoxyl-agarose and chitosan-glutaraldehyde derivatives were used to catalyze corncob xylan hydrolysis, reaching 72 % conversion, representing a xylose productivity of around 20 g L-1 h-1. After ten 4h-cycles (pH 6.0, 35 °C), the xylan-to-xylose conversion remained approximately unchanged. Therefore, the immobilized ß-xylosidases prepared in this work can be of great interest as biocatalysts in a biorefinery context.

Machine learning applied for metabolic flux-based control of micro-aerated fermentations in bioreactors.

Various bio-based processes depend on controlled micro-aerobic conditions to achieve a satisfactory product yield. However, the limiting oxygen concentration varies according to the micro-organism employed, while for industrial applications, there is no cost-effective way of measuring it at low levels. This study proposes a machine learning procedure within a metabolic flux-based control strategy (SUPERSYS_MCU) to address this issue. The control strategy used simulations of a genome-scale metabolic model to generate a surrogate model in the form of an artificial neural network, to be used in a micro-aerobic fermentation strategy (MF-ANN). The meta-model provided setpoints to the controller, allowing adjustment of the inlet air flow to control the oxygen uptake rate. The strategy was evaluated in micro-aerobic batch cultures employing industrial Saccharomyces cerevisiae yeast, with defined medium and glucose as the carbon source, as a case study. The performance of the proposed control scheme was compared with a conventional fermentation and with three previously reported micro-aeration strategies, including respiratory quotient-based control and constant air flow rate. Due to maintenance of the oxidative balance at the anaerobiosis threshold, the MF-ANN provided volumetric ethanol productivity of 4.16 g·L-1 ·h-1 and a yield of 0.48 gethanol .gsubstrate-1 , which were higher than the values achieved for the other conditions studied (maximum of 3.4 g·L-1 ·h-1 and 0.35-0.40 gethanol ·gsubstrate-1 , respectively). Due to its modular character, the MF-ANN strategy could be adapted to other micro-aerated bioprocesses.

High stabilization and hyperactivation of a recombinant β-Xylosidase through immobilization strategies

Attainment of a stable and highly active β-xylosidase is of major importance for the efficient and cost-competitive hydrolysis of hemicellulose xylan, as well as for its industrial conversion into biofuels and biochemicals. Here, a recombinant β-xylosidase of the glycoside hydrolase family (GH43) from Bacillus subtilis was produced in Escherichia coli culture, purified, and subsequently immobilized on agarose and chitosan. Glutaraldehyde and glyoxyl groups were evaluated as activating agents to select the most efficient derivative. Multi-point immobilization on agarose led to an extraordinary thermal stability (half-lives 3604 and 164-fold higher than the free enzyme, at 50° and 35 °C, respectively). Even for chitosan activated with glutaraldehyde, a low-cost support, thermal stability of the immobilized enzyme was 326 and 12-fold higher than the free enzyme at 50° and 35°C, respectively. Immobilized enzymes showed no release of any subunit for the agarose-glyoxyl derivative, and only a few ones for the support activated with glutaraldehyde. Most remarkably, the enzyme kinetic behavior after immobilization increased up to 4-fold in relation to the free one. β-xylosidase, a tetrameric enzyme with four identical subunits, exists in equilibrium between the monomeric and oligomeric forms in solution. Depending on the pH of immobilization, the enzyme oligomerization can be favored, thus explaining the hyperactivation phenomenon. Both glyoxyl-agarose and chitosan-glutaraldehyde derivatives were used to catalyze corncob xylan hydrolysis, reaching 72 % conversion, representing a xylose productivity of around 20 g L−1 h−1. After ten 4h-cycles (pH 6.0, 35 °C), the xylan-to-xylose conversion remained approximately unchanged. Therefore, the immobilized β-xylosidases prepared in this work can be of great interest as biocatalysts in a biorefinery context.

Repeated batches as a strategy for high 2G ethanol production from undetoxified hemicellulose hydrolysate using immobilized cells of recombinant Saccharomyces cerevisiae in a fixed-bed reactor.

BACKGROUND: The search for sustainable energy sources has become a worldwide issue, making the development of efficient biofuel production processes a priority. Immobilization of second-generation (2G) xylose-fermenting Saccharomyces cerevisiae strains is a promising approach to achieve economic viability of 2G bioethanol production from undetoxified hydrolysates through operation at high cell load and mitigation of inhibitor toxicity. In addition, the use of a fixed-bed reactor can contribute to establish an efficient process because of its distinct advantages, such as high conversion rate per weight of biocatalyst and reuse of biocatalyst. RESULTS: This work assessed the influence of alginate entrapment on the tolerance of recombinant S. cerevisiae to acetic acid. Encapsulated GSE16-T18SI.1 (T18) yeast showed an outstanding performance in repeated batch fermentations with cell recycling in YPX medium supplemented with 8 g/L acetic acid (pH 5.2), achieving 10 cycles without significant loss of productivity. In the fixed-bed bioreactor, a high xylose fermentation rate with ethanol yield and productivity values of 0.38 gethanol/gsugars and 5.7 g/L/h, respectively were achieved in fermentations using undetoxified sugarcane bagasse hemicellulose hydrolysate, with and without medium recirculation. CONCLUSIONS: The performance of recombinant strains developed for 2G ethanol production can be boosted strongly by cell immobilization in alginate gels. Yeast encapsulation allows conducting fermentations in repeated batch mode in fixed-bed bioreactors with high xylose assimilation rate and high ethanol productivity using undetoxified hemicellulose hydrolysate.

Cost analysis based on bioreactor cultivation conditions: Production of a soluble recombinant protein using Escherichia coli BL21(DE3).

The impact of cultivation strategy on the cost of recombinant protein production is crucial for defining cost-effective bioreactor operation conditions. This paper presents a methodology to estimate and compare cost impacts related to utilities as well as medium composition, using simple design equations and accessible data. Data from batch bioreactor cultures were used as case study involving the production of pneumococcal surface protein A, a soluble recombinant protein, employing E. coli BL21(DE3). Cultivation strategies and corresponding process costs covered a wide range of operational conditions, including different media, inducers, and temperatures. The core expenses were related to the medium and cooling. When the price of peptone was above the threshold value of US$ 30/kg, defined medium became the best choice. IPTG and temperatures around 32 °C led to shorter cultures and lower PspA4Pro production costs. The procedure offers a simple, accessible theoretical tool to identify cost-effective production strategies using bioreactors.

Modeling and simulation of anion exchange chromatography for purification of proteins in complex mixtures.

Ion exchange chromatography is extensively used in the purification of biological compounds. Reliable mathematical models describing this chromatographic technique are available and can be used to improve the performance of this separation step. However, the use of synthetic mixtures for model development hampers the application of this approach with real cell extracts processed in downstream operations. This work presents an original approach for handling non-synthetic genuine mixtures of proteins, which was applied in the purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro). First, evaluation was made of the efficiency of steric mass action (SMA) and modified Langmuir isotherms, which were separately used together with the equilibrium dispersive model (EDM). The data used for parameter estimation and model validation were obtained from anion exchange chromatography runs (employing Q-Sepharose FF), applied to real cell extracts produced by different cultivation strategies. Simulations showed that the models were able to describe the complex mixtures of unknown proteins. Next, the EDM and SMA approaches were used to separately describe the profile of PspA4Pro and the pool of protein impurities eluted together. The simulations showed that PspA4Pro tended to elute at the beginning of the peak, enabling the establishment of an alternative elution schedule that provided a 34% increase in the purity achieved using the anion exchange chromatography.

Modeling and simulation of anion exchange chromatography for purification of proteins in complex mixtures

Ion exchange chromatography is extensively used in the purification of biological compounds. Reliable mathematical models describing this chromatographic technique are available and can be used to improve the performance of this separation step. However, the use of synthetic mixtures for model development hampers the application of this approach with real cell extracts processed in downstream operations. This work presents an original approach for handling non-synthetic genuine mixtures of proteins, which was applied in the purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro). First, evaluation was made of the efficiency of steric mass action (SMA) and modified Langmuir isotherms, which were separately used together with the equilibrium dispersive model (EDM). The data used for parameter estimation and model validation were obtained from anion exchange chromatography runs (employing Q-Sepharose FF), applied to real cell extracts produced by different cultivation strategies. Simulations showed that the models were able to describe the complex mixtures of unknown proteins. Next, the EDM and SMA approaches were used to separately describe the profile of PspA4Pro and the pool of protein impurities eluted together. The simulations showed that PspA4Pro tended to elute at the beginning of the peak, enabling the establishment of an alternative elution schedule that provided a 34% increase in the purity achieved using the anion exchange chromatography.

Cost analysis based on bioreactor cultivation conditions: production of a soluble recombinant protein using Escherichia coli BL21(DE3)

The impact of cultivation strategy on the cost of recombinant protein production is crucial for defining cost-effective bioreactor operation conditions. This paper presents a methodology to estimate and compare cost impacts related to utilities as well as medium composition, using simple design equations and accessible data. Data from batch bioreactor cultures were used as case study involving the production of pneumococcal surface protein A, a soluble recombinant protein, employing E. coli BL21(DE3). Cultivation strategies and corresponding process costs covered a wide range of operational conditions, including different media, inducers, and temperatures. The core expenses were related to the medium and cooling. When the price of peptone was above the threshold value of US$ 30/kg, defined medium became the best choice. IPTG and temperatures around 32°C led to shorter cultures and lower PspA4Pro production costs. The procedure offers a simple, accessible theoretical tool to identify cost-effective production strategies using bioreactors.

Modeling and simulation of anion exchange chromatography for purification of proteins in complex mixtures

Ion exchange chromatography is extensively used in the purification of biological compounds. Reliable mathematical models describing this chromatographic technique are available and can be used to improve the performance of this separation step. However, the use of synthetic mixtures for model development hampers the application of this approach with real cell extracts processed in downstream operations. This work presents an original approach for handling non-synthetic genuine mixtures of proteins, which was applied in the purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro). First, evaluation was made of the efficiency of steric mass action (SMA) and modified Langmuir isotherms, which were separately used together with the equilibrium dispersive model (EDM). The data used for parameter estimation and model validation were obtained from anion exchange chromatography runs (employing Q-Sepharose FF), applied to real cell extracts produced by different cultivation strategies. Simulations showed that the models were able to describe the complex mixtures of unknown proteins. Next, the EDM and SMA approaches were used to separately describe the profile of PspA4Pro and the pool of protein impurities eluted together. The simulations showed that PspA4Pro tended to elute at the beginning of the peak, enabling the establishment of an alternative elution schedule that provided a 34% increase in the purity achieved using the anion exchange chromatography.

Cost analysis based on bioreactor cultivation conditions: production of a soluble recombinant protein using Escherichia coli BL21(DE3)

The impact of cultivation strategy on the cost of recombinant protein production is crucial for defining cost-effective bioreactor operation conditions. This paper presents a methodology to estimate and compare cost impacts related to utilities as well as medium composition, using simple design equations and accessible data. Data from batch bioreactor cultures were used as case study involving the production of pneumococcal surface protein A, a soluble recombinant protein, employing E. coli BL21(DE3). Cultivation strategies and corresponding process costs covered a wide range of operational conditions, including different media, inducers, and temperatures. The core expenses were related to the medium and cooling. When the price of peptone was above the threshold value of US$ 30/kg, defined medium became the best choice. IPTG and temperatures around 32°C led to shorter cultures and lower PspA4Pro production costs. The procedure offers a simple, accessible theoretical tool to identify cost-effective production strategies using bioreactors.

A hierarchical state estimation and control framework for monitoring and dissolved oxygen regulation in bioprocesses.

The integration of state estimation and control is a promising approach to overcome challenges related to unavailable or noisy online measurements and plant-model mismatch. Extended Kalman filter (EKF) and moving horizon estimator (MHE) are widely used methods that have complementary features. EKF provides fast estimation and MHE optimal performance. In this paper, a novel hierarchical EKF/MHE approach combined with a dynamic matrix controller (DMC), denoted as EKF/MHE-DMC, is proposed for process monitoring and dissolved oxygen control in airlift bioreactors. The approach is successfully tested in simulated cultivations of Escherichia coli for recombinant protein production, considering specific scenarios of step set point tracking, step disturbance rejection, plant-model mismatch, and measurement noise. Results also show that, given a model that describes the measured dissolved oxygen precisely, as assumed in this study for the in silico experiments, the EKF/MHE-DMC approach is able to estimate the cell, protein, substrate, and dissolved oxygen concentrations based only on the measurement of the latter, reducing the estimation error by 93.8% when compared to a benchmark case employing EKF and DMC. The general structure of the proposed EKF/MHE-DMC framework could be adapted for implementation on other relevant bioprocess systems employing their derived process models.

Mapping Salmonella typhimurium pathways using 13C metabolic flux analysis.

In the last years, Salmonella has been extensively studied not only due to its importance as a pathogen, but also as a host to produce pharmaceutical compounds. However, the full exploitation of Salmonella as a platform for bioproduct delivery has been hampered by the lack of information about its metabolism. Genome-scale metabolic models can be valuable tools to delineate metabolic engineering strategies as long as they closely represent the actual metabolism of the target organism. In the present study, a 13C-MFA approach was applied to map the fluxes at the central carbon pathways of S. typhimurium LT2 growing at glucose-limited chemostat cultures. The experiments were carried out in a 2L bioreactor, using defined medium enriched with 20% 13C-labeled glucose. Metabolic flux distributions in central carbon pathways of S. typhimurium LT2 were estimated using OpenFLUX2 based on the labeling pattern of biomass protein hydrolysates together with biomass composition. The results suggested that pentose phosphate is used to catabolize glucose, with minor fluxes through glycolysis. In silico simulations, using Optflux and pFBA as simulation method, allowed to study the performance of the genome-scale metabolic model. In general, the accuracy of in silico simulations was improved by the superimposition of estimated intracellular fluxes to the existing genome-scale metabolic model, showing a better fitting to the experimental extracellular fluxes, whereas the intracellular fluxes of pentose phosphate and anaplerotic reactions were poorly described.

Production and purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro) with high-purity and low endotoxin content.

Streptococcus pneumoniae is the main cause of pneumonia, meningitis, and other conditions that kill thousands of children every year worldwide. The replacement of pneumococcal serotypes among the vaccinated population has evidenced the need for new vaccines with broader coverage and driven the research for protein-based vaccines. Pneumococcal surface protein A (PspA) protects S. pneumoniae from the bactericidal effect of human apolactoferrin and prevents complement deposition. Several studies indicate that PspA is a very promising target for novel vaccine formulations. Here we describe a production and purification process for an untagged recombinant fragment of PspA from clade 4 (PspA4Pro), which has been shown to be cross-reactive with several PspA variants. PspA4Pro was obtained using lactose as inducer in Phytone auto-induction batch or glycerol limited fed-batch in 5-L bioreactor. The purification process includes two novel steps: (i) clarification using a cationic detergent to precipitate contaminant proteins, nucleic acids, and other negatively charged molecules as the lipopolysaccharide, which is the major endotoxin; and (ii) cryoprecipitation that eliminates aggregates and contaminants, which precipitate at -20 °C and pH 4.0, leaving PspA4Pro in the supernatant. The final process consisted of cell rupture in a continuous high-pressure homogenizer, clarification, anion exchange chromatography, cryoprecipitation, and cation exchange chromatography. This process avoided costly tag removal steps and recovered 35.3 ± 2.5% of PspA4Pro with 97.8 ± 0.36% purity and reduced endotoxin concentration by >99.9%. Circular dichroism and lactoferrin binding assay showed that PspA4Pro secondary structure and biological activity were preserved after purification and remained stable in a wide range of temperatures and pH values.