This document describes a study that applied a fed-batch bioprocess to produce the human soluble catechol-O-methyltransferase (hSCOMT) protein in Escherichia coli. A kinetic model was developed to predict biomass, substrate, and product concentrations over time based on data from fed-batch experiments with different glycerol feeding profiles. The model distinguished between an initial cell growth phase and a subsequent protein production phase after induction. The model was able to satisfactorily predict experimental results using a limited amount of data, without requiring exhaustive process knowledge. A validation experiment showed the model could accurately extrapolate to new conditions.
This document discusses various metabolic engineering approaches to modify crop plants. It covers engineering pathways to increase production of desirable compounds like vitamins and minerals, and reduce antinutrients. Specific examples discussed include producing provitamin A in golden rice, modifying starch and fructan biosynthesis to improve gut health, and preventing cyanogenic glucoside production in cassava. Both transgenic and non-transgenic methods are described, including using genes only from within a crop's gene pool. The goal is to enhance crop productivity and nutritional quality for human health.
This document provides an overview of metabolic engineering. It defines metabolic engineering as the alteration of genetic components or flux in an organism to achieve a desired phenotype or product through modifying biochemical reactions or introducing new pathways using recombinant DNA technology. The history section outlines some early work in metabolic engineering in the 1980s-1990s, including modifying bacteria to produce various chemicals and improving growth properties. The document also discusses various approaches that have been developed for metabolic engineering, such as global transcription machinery engineering and using various omics techniques to analyze cellular processes.
This document describes an in silico platform developed to identify feasible heterologous pathways for producing non-native target metabolites in host microorganisms. The platform screens heterologous pathways by sequentially adding metabolic reactions from a database to expand the metabolic networks of Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae. It then uses flux balance analysis to estimate production yields and identify pathways associated with maximum growth rates of the host. The goal is to provide rational heterologous pathways and host microorganisms for efficient production of desired target metabolites.
key application of plant metabolic engineering Faisal Ali
This document discusses key applications of plant metabolic engineering, including enabling plants to fix their own nitrogen, altering nutrient content of crop plants, enhancing photosynthetic efficiency, and using plants for biofuel production. Some of the challenges discussed are expressing the large number of genes required for nitrogen fixation in plants, developing efficient nutrient exchange in plant-microbe symbiosis, and modifying lignin in plants to more easily access cellulose for biofuels while avoiding negative impacts on growth.
- Researchers identified over 700 plasma membrane proteins in Arabidopsis including members of the TPLATE complex required for endocytosis and various cell wall biosynthetic proteins. The free-flow electrophoresis approach purified plasma membrane vesicles with few contaminants.
- Overexpression of the rice wall-associated kinase OsWAK25 conferred resistance to two bacterial pathogens and increased susceptibility to two fungal pathogens. Overexpression of a related protein compromised resistance mediated by OsWAK25.
- Laboratory evolution of S. cerevisiae yielded a mutant glucose transporter, Hxt7(F79S), that improved growth on and uptake of xylose, enabling improved utilization of both sugars in biomass.
This document discusses metabolic engineering. Metabolic engineering modifies cellular properties through recombinant DNA technology to alter metabolic pathways for production of chemicals, fuels, pharmaceuticals and medicine. It requires overexpression or downregulation of proteins in pathways so cells produce new products. The first step is understanding the host cell environment for genetic modifications, and the effect of modifications on growth should be examined. Genetic manipulation may negatively impact metabolic burden. Metabolic engineering is used to produce various compounds through methods like eliminating competing pathways, expressing foreign enzymes, and optimizing cofactors like NAD+/NADH.
This document describes the development of a novel growth factor delivery system using poly(lactic-co-glycolic acid) (PLGA) microparticles. The inclusion of a hydrophilic PLGA-PEG-PLGA triblock copolymer alters the release kinetics from the microparticles such that growth factor release can occur before polymer degradation. Three formulations are identified as promising candidates for delivering growth factors like BMP-2, with adjustable release profiles from 4 days to over 4 weeks. Mixing microparticles of different formulations provides another method to control release kinetics. This customized, localized delivery system has the potential to improve the efficacy and safety of recombinant growth factor therapies.
This document discusses various metabolic engineering approaches to modify crop plants. It covers engineering pathways to increase production of desirable compounds like vitamins and minerals, and reduce antinutrients. Specific examples discussed include producing provitamin A in golden rice, modifying starch and fructan biosynthesis to improve gut health, and preventing cyanogenic glucoside production in cassava. Both transgenic and non-transgenic methods are described, including using genes only from within a crop's gene pool. The goal is to enhance crop productivity and nutritional quality for human health.
This document provides an overview of metabolic engineering. It defines metabolic engineering as the alteration of genetic components or flux in an organism to achieve a desired phenotype or product through modifying biochemical reactions or introducing new pathways using recombinant DNA technology. The history section outlines some early work in metabolic engineering in the 1980s-1990s, including modifying bacteria to produce various chemicals and improving growth properties. The document also discusses various approaches that have been developed for metabolic engineering, such as global transcription machinery engineering and using various omics techniques to analyze cellular processes.
This document describes an in silico platform developed to identify feasible heterologous pathways for producing non-native target metabolites in host microorganisms. The platform screens heterologous pathways by sequentially adding metabolic reactions from a database to expand the metabolic networks of Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae. It then uses flux balance analysis to estimate production yields and identify pathways associated with maximum growth rates of the host. The goal is to provide rational heterologous pathways and host microorganisms for efficient production of desired target metabolites.
key application of plant metabolic engineering Faisal Ali
This document discusses key applications of plant metabolic engineering, including enabling plants to fix their own nitrogen, altering nutrient content of crop plants, enhancing photosynthetic efficiency, and using plants for biofuel production. Some of the challenges discussed are expressing the large number of genes required for nitrogen fixation in plants, developing efficient nutrient exchange in plant-microbe symbiosis, and modifying lignin in plants to more easily access cellulose for biofuels while avoiding negative impacts on growth.
- Researchers identified over 700 plasma membrane proteins in Arabidopsis including members of the TPLATE complex required for endocytosis and various cell wall biosynthetic proteins. The free-flow electrophoresis approach purified plasma membrane vesicles with few contaminants.
- Overexpression of the rice wall-associated kinase OsWAK25 conferred resistance to two bacterial pathogens and increased susceptibility to two fungal pathogens. Overexpression of a related protein compromised resistance mediated by OsWAK25.
- Laboratory evolution of S. cerevisiae yielded a mutant glucose transporter, Hxt7(F79S), that improved growth on and uptake of xylose, enabling improved utilization of both sugars in biomass.
This document discusses metabolic engineering. Metabolic engineering modifies cellular properties through recombinant DNA technology to alter metabolic pathways for production of chemicals, fuels, pharmaceuticals and medicine. It requires overexpression or downregulation of proteins in pathways so cells produce new products. The first step is understanding the host cell environment for genetic modifications, and the effect of modifications on growth should be examined. Genetic manipulation may negatively impact metabolic burden. Metabolic engineering is used to produce various compounds through methods like eliminating competing pathways, expressing foreign enzymes, and optimizing cofactors like NAD+/NADH.
This document describes the development of a novel growth factor delivery system using poly(lactic-co-glycolic acid) (PLGA) microparticles. The inclusion of a hydrophilic PLGA-PEG-PLGA triblock copolymer alters the release kinetics from the microparticles such that growth factor release can occur before polymer degradation. Three formulations are identified as promising candidates for delivering growth factors like BMP-2, with adjustable release profiles from 4 days to over 4 weeks. Mixing microparticles of different formulations provides another method to control release kinetics. This customized, localized delivery system has the potential to improve the efficacy and safety of recombinant growth factor therapies.
JBEI Research Highlights - November 2017 Irina Silva
This document summarizes the development and testing of a quorum sensing (QS)-mediated gene expression system to control bisabolene production in engineered E. coli. Researchers developed a QS system using the LuxI/R genes from Vibrio fischeri to induce expression of the bisabolene production pathway without the need for external inducers. The best QS strain, with the sensor genes integrated into the genome and an optimized response plasmid, produced 1.1 g/L of bisabolene, a 44% improvement over previous inducible systems. This QS-based system provided defined and homogeneous gene expression and production compared to inducible controls.
Long-term conservation tillage practices over 37 years increased soil organic carbon and total nitrogen concentrations in surface soils compared to conventional tillage. Conservation tillage led to significant stratification of carbon and nitrogen in the soil profile. The loamy sand soils approached carbon saturation after approximately 40 years under conservation tillage combined with cover crops, demonstrating the potential for carbon sequestration in cropland soils with these management practices.
This document discusses using a chemical genetics approach to study lipid accumulation and cell growth regulation in the algae C. reinhardtii. The goal is to identify chemical and genetic modulators of these processes to maximize algal lipid yields for biodiesel production. The proposed approach involves high-throughput screening of compound libraries to identify molecules that trigger lipid accumulation or growth stimulation. Target identification strategies will then determine the proteins targeted by the most effective molecules to uncover regulatory genes involved. This could enable direct use of chemical modulators or genetic engineering of regulatory pathways to optimize algal metabolism for biodiesel production.
This study assessed the enzymatic hydrolysis of proteins from the microalgae Chlorella pyrenoidosa and Spirulina sp. LEB 18 to produce protein hydrolysates. Three commercial proteases were used under different conditions to hydrolyze the microalgae proteins. The highest degrees of hydrolysis for Spirulina and Chlorella, respectively, were 55.31% and 52.9% and were obtained with 4 hours of reaction time using Protemax N200 protease. Statistical analysis showed that enzyme concentration, substrate concentration, and reaction time significantly affected the degree of hydrolysis. The results indicate it is possible to obtain protein hydrolysates with varying degrees of hydrolysis from microalgae
High Throughput Screening for Glycogen and PolyglucosanBen Decker
This document discusses high throughput screening (HTS) for identifying compounds that modulate glycogen and polyglucosan. HTS uses robotics, liquid handling devices, and detectors to quickly test thousands of compounds. The researcher has prepared an assay to observe changes in glycogen quality and quantity using limited digestion and staining. Mouse fibroblasts with genetic defects resulting in low glycogen branching enzyme activity and no polyglucosan are being used. Currently, 1700 FDA-approved compounds are being tested in triplicates using an automated assay to identify potential modulators of glycogen and polyglucosan pathways.
The document describes a study that screened 168 yeast strains for tolerance to the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2Ciim][OAc]). 13 strains were found to be tolerant to 5% [C2Ciim][OAc], with Galactomyces geotrichum being the most tolerant. G. geotrichum exhibited enhanced growth in the ionic liquid medium. Several yeasts were also identified that were capable of rapid growth and high cell density in 5% of the ionic liquid.
1. A microfluidic chip was developed that can integrate DNA assembly and bacterial transformation through electroporation in droplets. Multiple assembly methods like Golden Gate, Gibson, and yeast assembly were implemented on-chip with 95% sequence matching to conventional methods using much less reagents.
2. The draft genome of Raoultella terrigena, a nitrogen-fixing endophytic bacterium isolated from tobacco roots, was sequenced. It contains genes for nitrogen fixation and for producing plant hormones and volatiles that promote plant growth. Understanding its genomics may help develop bioenergy crops requiring less inputs.
3. A study analyzed the arbuscular mycorrhizal fungi communities associated with the roots of black
Metabolic engineering approaches in medicinal plantsN Poorin
1) Metabolic engineering approaches aim to enhance production of valuable secondary metabolites in medicinal plants by manipulating metabolic pathways through techniques like overexpressing or suppressing genes.
2) A case study demonstrated enhancing taxadiene production in yew trees by suppressing competing carotenoid pathways. Another case study used integrated metabolomic and transcriptomic analysis to map metabolic pathways producing terpenoid indole alkaloids in Catharanthus roseus.
3) Metabolic engineering approaches include introducing multigene constructs to simultaneously modify multiple steps in a pathway, as well as combining pathway enzymes to generate novel decorated metabolites. However, challenges remain around fully characterizing plant metabolic networks.
The document describes research that identified the first enzyme capable of producing toluene through a biochemical reaction. Key findings include:
- Researchers successfully identified the enzyme phenylacetate decarboxylase (PhdB) and its activating enzyme (PhdA) that enable the production of toluene from microbial communities.
- This is the first reported enzyme for toluene biosynthesis and expands the catalytic abilities of glycyl radical enzymes.
- The discovery will allow the first biochemical synthesis of renewable toluene from lignocellulosic biomass rather than petroleum, offering potential for biofuel production.
JBEI Research Highlights - November 2018 Irina Silva
This document discusses recent advances in x-ray hydroxyl radical footprinting at the Advanced Light Source synchrotron. It compares dose response curves and mass spectrometry results from focused and unfocused white light sources. It also describes developing "drop-on-demand" methodologies to increase sample dose while maintaining microsecond exposure times, which enables high-dose experiments while minimizing sample volume. Preliminary experiments demonstrate this approach yields high quality data. The document contributes to improving synchrotron hydroxyl radical footprinting techniques for investigating protein and nucleic acid structures.
JBEI Research Highlights - October 2017 Irina Silva
This document summarizes four research articles:
1) The first describes developing a library of hybrid promoters in yeast that can be induced to different levels using the same synthetic transcription factor and estradiol.
2) The second presents ClusterCAD, a computational tool for designing chimeric polyketide synthases to enable combinatorial biosynthesis of bioproducts.
3) The third introduces multiple marker abundance profiling (MMAP) to estimate organelle abundance from protein identifiers over a range of conditions.
4) The fourth demonstrates improving membrane protein expression and function in E. coli using genomic edits identified through a transposon library screen.
- The document discusses four types of forage sorghum (brown-midrib, non-brown-midrib, photoperiod sensitive, and photoperiod insensitive) and their potential impact on the minimum ethanol selling price at a biorefinery using ionic liquid pretreatment.
- Simulation results show that non-photoperiod sensitive sorghum may result in lower cost biofuels compared to high-yielding photoperiod sensitive varieties under certain conditions.
- If lignin value increases, non-brown-midrib varieties become the most attractive option due to their higher biomass yield outweighing the lower lignin content of brown-midrib varieties.
This document discusses plant metabolic engineering. It begins with an introduction to metabolic engineering and explains that it involves modifying biochemical reactions or introducing new ones using recombinant DNA to direct the formation of products or modify cellular properties. It then discusses plants as natural factories for metabolic engineering due to their low cost and ease of growth. Various strategies for plant metabolic engineering are covered, including modifying rate limiting steps or diverting flux. Applications like producing antibodies, edible vaccines, polymers and pharmaceuticals in plants are also summarized. Emerging technologies and the future of the field are then outlined.
This study evaluated methods for preserving microbial communities enriched on switchgrass biomass. High-throughput sequencing showed the communities shifted during initial enrichment but remained active after storage. Storage in liquid nitrogen without cryoprotectants resulted in variable preservation of dominant microbes, while cryopreservation with DMSO or glycerol provided consistent preservation. Despite shifts in relative abundance, the preserved samples retained an active community, suggesting the presence rather than abundance of microbes is important for activity.
Recent advances in technoeconomic analysis (TEA) were reviewed:
- TEA is useful for process design, cost estimation, and identifying bottlenecks early in research.
- Studies now enable faster iteration, robust uncertainty analysis, and open-source platforms.
- Trends include more expansive system boundaries and potential integration with high-throughput experiments.
The document proposes developing idealized versions of bioenergy crops, called ideotypes, to guide bioengineering efforts. Through a literature review, the authors synthesize approaches for engineering different feedstock crops to be tailored for specific biomass or bioproduct production and accumulation. They suggest how crops can be engineered for the production and accumulation of different types of biomass or specific bioproducts to make bioenergy crops more economically and environmentally favorable.
This study aimed to improve olefin tolerance and production in E. coli. The researchers identified that the AcrAB-TolC efflux pump is involved in styrene and 1-hexene tolerance. For styrene, the pump was essential for optimal production but hindered improvement efforts. For 1-hexene, pump variants were evolved that improved tolerance. The variants had up to six beneficial mutations localized on the AcrB trimer structure, demonstrating efflux pumps are an important target for improving chemical tolerance and production optimization.
- The study investigated using arbuscular mycorrhizal fungi (AMF) colonization and tree-herb interactions to enhance growth of legume trees for biofuel production on lead contaminated soil.
- AMF inoculation of legume trees increased overall biomass yield compared to non-inoculated plants under lead stress, likely by retaining lead in underground parts and improving nutrient acquisition.
- Combining AMF with planting legume trees with legume or grass herbs further optimized the system for phytoremediation of contaminated marginal lands and production of biofuel feedstocks.
White-footed ants feed on plant nectar and honeydew and reproduce through budding, forming large colonies of up to 3 million ants nested in trees, bushes, and structures. Crazy ants form populous colonies with many queens and workers that occur in temporary, mobile nests. Ghost ants occupy multiple nest sites connected by odor trails and will move nests when disturbed. Pavement ants form large colonies through nuptial flights and egg/larval development takes 6-9 weeks. Big-headed ants form massive, interconnected supercolonies through budding and multiple queens. Carpenter ants develop through complete metamorphosis and found satellite nests near mature colonies.
Norway has a long history of aquaculture dating back to the 1850s. Today, salmon and rainbow trout farming have developed into major industries along Norway's coast utilizing ocean cage systems. Atlantic salmon and rainbow trout account for the majority of production. Other species being farmed include cod, halibut, spotted wolffish, Arctic charr, oysters, mussels, and sea urchins. Farming practices involve breeding programs, hatcheries, ongrowing in ocean cages, and processing facilities. Aquaculture has grown significantly and brought economic benefits, though employment levels have decreased as operations have become more efficient.
JBEI Research Highlights - November 2017 Irina Silva
This document summarizes the development and testing of a quorum sensing (QS)-mediated gene expression system to control bisabolene production in engineered E. coli. Researchers developed a QS system using the LuxI/R genes from Vibrio fischeri to induce expression of the bisabolene production pathway without the need for external inducers. The best QS strain, with the sensor genes integrated into the genome and an optimized response plasmid, produced 1.1 g/L of bisabolene, a 44% improvement over previous inducible systems. This QS-based system provided defined and homogeneous gene expression and production compared to inducible controls.
Long-term conservation tillage practices over 37 years increased soil organic carbon and total nitrogen concentrations in surface soils compared to conventional tillage. Conservation tillage led to significant stratification of carbon and nitrogen in the soil profile. The loamy sand soils approached carbon saturation after approximately 40 years under conservation tillage combined with cover crops, demonstrating the potential for carbon sequestration in cropland soils with these management practices.
This document discusses using a chemical genetics approach to study lipid accumulation and cell growth regulation in the algae C. reinhardtii. The goal is to identify chemical and genetic modulators of these processes to maximize algal lipid yields for biodiesel production. The proposed approach involves high-throughput screening of compound libraries to identify molecules that trigger lipid accumulation or growth stimulation. Target identification strategies will then determine the proteins targeted by the most effective molecules to uncover regulatory genes involved. This could enable direct use of chemical modulators or genetic engineering of regulatory pathways to optimize algal metabolism for biodiesel production.
This study assessed the enzymatic hydrolysis of proteins from the microalgae Chlorella pyrenoidosa and Spirulina sp. LEB 18 to produce protein hydrolysates. Three commercial proteases were used under different conditions to hydrolyze the microalgae proteins. The highest degrees of hydrolysis for Spirulina and Chlorella, respectively, were 55.31% and 52.9% and were obtained with 4 hours of reaction time using Protemax N200 protease. Statistical analysis showed that enzyme concentration, substrate concentration, and reaction time significantly affected the degree of hydrolysis. The results indicate it is possible to obtain protein hydrolysates with varying degrees of hydrolysis from microalgae
High Throughput Screening for Glycogen and PolyglucosanBen Decker
This document discusses high throughput screening (HTS) for identifying compounds that modulate glycogen and polyglucosan. HTS uses robotics, liquid handling devices, and detectors to quickly test thousands of compounds. The researcher has prepared an assay to observe changes in glycogen quality and quantity using limited digestion and staining. Mouse fibroblasts with genetic defects resulting in low glycogen branching enzyme activity and no polyglucosan are being used. Currently, 1700 FDA-approved compounds are being tested in triplicates using an automated assay to identify potential modulators of glycogen and polyglucosan pathways.
The document describes a study that screened 168 yeast strains for tolerance to the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2Ciim][OAc]). 13 strains were found to be tolerant to 5% [C2Ciim][OAc], with Galactomyces geotrichum being the most tolerant. G. geotrichum exhibited enhanced growth in the ionic liquid medium. Several yeasts were also identified that were capable of rapid growth and high cell density in 5% of the ionic liquid.
1. A microfluidic chip was developed that can integrate DNA assembly and bacterial transformation through electroporation in droplets. Multiple assembly methods like Golden Gate, Gibson, and yeast assembly were implemented on-chip with 95% sequence matching to conventional methods using much less reagents.
2. The draft genome of Raoultella terrigena, a nitrogen-fixing endophytic bacterium isolated from tobacco roots, was sequenced. It contains genes for nitrogen fixation and for producing plant hormones and volatiles that promote plant growth. Understanding its genomics may help develop bioenergy crops requiring less inputs.
3. A study analyzed the arbuscular mycorrhizal fungi communities associated with the roots of black
Metabolic engineering approaches in medicinal plantsN Poorin
1) Metabolic engineering approaches aim to enhance production of valuable secondary metabolites in medicinal plants by manipulating metabolic pathways through techniques like overexpressing or suppressing genes.
2) A case study demonstrated enhancing taxadiene production in yew trees by suppressing competing carotenoid pathways. Another case study used integrated metabolomic and transcriptomic analysis to map metabolic pathways producing terpenoid indole alkaloids in Catharanthus roseus.
3) Metabolic engineering approaches include introducing multigene constructs to simultaneously modify multiple steps in a pathway, as well as combining pathway enzymes to generate novel decorated metabolites. However, challenges remain around fully characterizing plant metabolic networks.
The document describes research that identified the first enzyme capable of producing toluene through a biochemical reaction. Key findings include:
- Researchers successfully identified the enzyme phenylacetate decarboxylase (PhdB) and its activating enzyme (PhdA) that enable the production of toluene from microbial communities.
- This is the first reported enzyme for toluene biosynthesis and expands the catalytic abilities of glycyl radical enzymes.
- The discovery will allow the first biochemical synthesis of renewable toluene from lignocellulosic biomass rather than petroleum, offering potential for biofuel production.
JBEI Research Highlights - November 2018 Irina Silva
This document discusses recent advances in x-ray hydroxyl radical footprinting at the Advanced Light Source synchrotron. It compares dose response curves and mass spectrometry results from focused and unfocused white light sources. It also describes developing "drop-on-demand" methodologies to increase sample dose while maintaining microsecond exposure times, which enables high-dose experiments while minimizing sample volume. Preliminary experiments demonstrate this approach yields high quality data. The document contributes to improving synchrotron hydroxyl radical footprinting techniques for investigating protein and nucleic acid structures.
JBEI Research Highlights - October 2017 Irina Silva
This document summarizes four research articles:
1) The first describes developing a library of hybrid promoters in yeast that can be induced to different levels using the same synthetic transcription factor and estradiol.
2) The second presents ClusterCAD, a computational tool for designing chimeric polyketide synthases to enable combinatorial biosynthesis of bioproducts.
3) The third introduces multiple marker abundance profiling (MMAP) to estimate organelle abundance from protein identifiers over a range of conditions.
4) The fourth demonstrates improving membrane protein expression and function in E. coli using genomic edits identified through a transposon library screen.
- The document discusses four types of forage sorghum (brown-midrib, non-brown-midrib, photoperiod sensitive, and photoperiod insensitive) and their potential impact on the minimum ethanol selling price at a biorefinery using ionic liquid pretreatment.
- Simulation results show that non-photoperiod sensitive sorghum may result in lower cost biofuels compared to high-yielding photoperiod sensitive varieties under certain conditions.
- If lignin value increases, non-brown-midrib varieties become the most attractive option due to their higher biomass yield outweighing the lower lignin content of brown-midrib varieties.
This document discusses plant metabolic engineering. It begins with an introduction to metabolic engineering and explains that it involves modifying biochemical reactions or introducing new ones using recombinant DNA to direct the formation of products or modify cellular properties. It then discusses plants as natural factories for metabolic engineering due to their low cost and ease of growth. Various strategies for plant metabolic engineering are covered, including modifying rate limiting steps or diverting flux. Applications like producing antibodies, edible vaccines, polymers and pharmaceuticals in plants are also summarized. Emerging technologies and the future of the field are then outlined.
This study evaluated methods for preserving microbial communities enriched on switchgrass biomass. High-throughput sequencing showed the communities shifted during initial enrichment but remained active after storage. Storage in liquid nitrogen without cryoprotectants resulted in variable preservation of dominant microbes, while cryopreservation with DMSO or glycerol provided consistent preservation. Despite shifts in relative abundance, the preserved samples retained an active community, suggesting the presence rather than abundance of microbes is important for activity.
Recent advances in technoeconomic analysis (TEA) were reviewed:
- TEA is useful for process design, cost estimation, and identifying bottlenecks early in research.
- Studies now enable faster iteration, robust uncertainty analysis, and open-source platforms.
- Trends include more expansive system boundaries and potential integration with high-throughput experiments.
The document proposes developing idealized versions of bioenergy crops, called ideotypes, to guide bioengineering efforts. Through a literature review, the authors synthesize approaches for engineering different feedstock crops to be tailored for specific biomass or bioproduct production and accumulation. They suggest how crops can be engineered for the production and accumulation of different types of biomass or specific bioproducts to make bioenergy crops more economically and environmentally favorable.
This study aimed to improve olefin tolerance and production in E. coli. The researchers identified that the AcrAB-TolC efflux pump is involved in styrene and 1-hexene tolerance. For styrene, the pump was essential for optimal production but hindered improvement efforts. For 1-hexene, pump variants were evolved that improved tolerance. The variants had up to six beneficial mutations localized on the AcrB trimer structure, demonstrating efflux pumps are an important target for improving chemical tolerance and production optimization.
- The study investigated using arbuscular mycorrhizal fungi (AMF) colonization and tree-herb interactions to enhance growth of legume trees for biofuel production on lead contaminated soil.
- AMF inoculation of legume trees increased overall biomass yield compared to non-inoculated plants under lead stress, likely by retaining lead in underground parts and improving nutrient acquisition.
- Combining AMF with planting legume trees with legume or grass herbs further optimized the system for phytoremediation of contaminated marginal lands and production of biofuel feedstocks.
White-footed ants feed on plant nectar and honeydew and reproduce through budding, forming large colonies of up to 3 million ants nested in trees, bushes, and structures. Crazy ants form populous colonies with many queens and workers that occur in temporary, mobile nests. Ghost ants occupy multiple nest sites connected by odor trails and will move nests when disturbed. Pavement ants form large colonies through nuptial flights and egg/larval development takes 6-9 weeks. Big-headed ants form massive, interconnected supercolonies through budding and multiple queens. Carpenter ants develop through complete metamorphosis and found satellite nests near mature colonies.
Norway has a long history of aquaculture dating back to the 1850s. Today, salmon and rainbow trout farming have developed into major industries along Norway's coast utilizing ocean cage systems. Atlantic salmon and rainbow trout account for the majority of production. Other species being farmed include cod, halibut, spotted wolffish, Arctic charr, oysters, mussels, and sea urchins. Farming practices involve breeding programs, hatcheries, ongrowing in ocean cages, and processing facilities. Aquaculture has grown significantly and brought economic benefits, though employment levels have decreased as operations have become more efficient.
Sighting of cockroaches can be distressing, as they are associated with filth. Learn more about their behavioural biology and commonly found in Malaysia, to protect your home and family.
This document summarizes past studies that have compared automakers' pre-regulatory cost estimates for complying with emission standards to the actual costs incurred. It finds that automakers have historically overestimated costs by 2-10 times, while regulators' estimates are closer but still often overestimated. Unanticipated technological innovation has dramatically lowered actual compliance costs. Based on this pattern, the document argues that California's new CO2 standards for automobiles will likely have similar nationwide impact at costs consistent with or lower than regulators' estimates due to innovation.
Keynote talk given at Fairdom User meeting http://fair-dom.org/communities/users/barcelona-2016-first-user-meeting/ .
I begin by summarising how we apply molecular approaches to understand social behaviour in ants. Subsequently, I give an overview of the data-handling challenges the genomic bioinformatics community faces. Finally, I give an overview of some of the tools and approaches my lab have developed to help us get things done better, faster, more reliably and more reproducibly.
1) Glyphosate resistance has emerged in over 20 weed species worldwide through various mechanisms including target site mutations and increased metabolism.
2) Target site mutations in the EPSPS gene have been identified in several glyphosate resistant weed species resulting in 2-3X levels of resistance.
3) Metabolic resistance allows some weed species to break down glyphosate faster through enhanced neutralizing enzyme activity, resulting in weaker resistance.
Fire ant stings: pathophysiology and natural remedyKevin KF Ng
Fire ant stings cause local inflammation and pain through the action of venom components. In rare cases, they can cause systemic allergic reactions. Current topical treatments are limited because they target only a few inflammatory mediators. The document proposes that phytochemicals extracted from plants may provide a more complete treatment by inhibiting multiple mediators of inflammation. Experimental evidence shows that a phytochemical preparation reduced inflammation from fire ant stings better than untreated controls. The conclusion is that phytochemicals offer a safe, effective, and inexpensive way to update the treatment of fire ant stings.
Fire ants, (solenopsis invicta), dry and store pieces of insect for later use...Vivi Yunisa
The document discusses how fire ant colonies store food. It found that fire ants collect insect prey and transform it into small dried pieces (jerky) in their nest. The ants transport solid food pieces in their mandibles but take liquid foods into their crop and distribute it through trophallaxis. This separation of solid and liquid food streams in their collection and storage may be evolutionarily advantageous.
This document discusses several types of cockroaches: American, brown banded, German, oriental, and smoky brown. It covers their life cycles and key identifying features to distinguish between the different cockroach species.
The document provides information about tramp ant species and the Rasberry crazy ant. It discusses the characteristics of tramp ants that allow them to successfully invade new areas, including being omnivorous, having multiple queens and nests, and dispersing via budding. It then focuses on the Rasberry crazy ant, providing details on its appearance, life cycle, and colony behavior. The document expresses concerns about the ant's potential spread and impacts, and recommends prevention and treatment methods like using Termidor as a barrier and Advance Carpenter Ant bait.
This document provides an overview of current regulatory strategies for developing biotherapeutic products. It discusses defining biotherapeutics, key challenges in their development including complex manufacturing processes in living systems, and strategies for cell banking, viral safety, and managing heterogeneity. The presentation emphasizes the importance of close communication with health authorities, using scientific advice and comparability assessments to bridge data from pre- and post-process changes. Maintaining consistent quality, safety and efficacy while making multiple necessary changes requires characterization, control of impurities, and linking manufacturing operations to critical quality attributes.
This document discusses research into the sociogenetics of fire ants. It describes experiments conducted to identify genes that change activity levels in orphaned young fire ant queens over time after being separated from their colony. RNA was extracted from queens at 0, 6, and 24 hours after orphaning and hybridized to microarrays containing 10,000 ant genes. Analysis found significant changes in the activity of 297 genes, most occurring 24 hours after orphaning. The goal is to understand how orphaning affects gene expression and physiological changes in young queens.
RNA silencing/RNAi involves the knock-down of genes through two types of small RNA molecules, miRNAs and siRNAs, that are involved in post-transcriptional and transcriptional gene silencing as an antiviral mechanism; short double-stranded RNAs are processed by the Dicer enzyme into siRNAs which are incorporated into the RISC complex to guide degradation of homologous mRNA targets; RNAi is an important endogenous gene regulatory mechanism and has applications in gene function analysis, gene therapy, and cancer treatment.
STRUCTURAL ORGANISATIONS IN ANIMALS
COCKROACH
The slide contain about-
1. Classification of cockroach
2. Feature of cockroach
3. Morphology- Head , Thorax , Abdomen
4. Difference between male and female cockroach
5. Anatomy- Digestive system, Blood vascular system, Respiratory system , Excretion , Nervous system , Reproductive system , Questions with answers
RNA Interference(RNAi) is a conserved biological response of eukaryotes against double-stranded RNA causing silencing of the gene expression. This mechanism has an important role in defending cells against viral genes and transposons. RNAi technology has become the latest "next big thing," progressing from a barely understood colour silencing mechanism found in flowers to a powerful tool that is going to become a new therapeutic tool for treating illnesses ranging from AIDS to cancer to Huntington’s disease. Even more exciting is the potential of RNAi in agriculture where it has provided a way to control pests and diseases as well as increase nutritional value of food.
The poem describes the movements of a cockroach in a room and how the poet sees similarities between the cockroach's restless behavior and human nature. The cockroach paces around the room, tracing patterns on the floor and flipping over objects, growing more agitated over time as it seems uncertain of where to go. In the final line, the poet recognizes something of himself in the cockroach's movements, using it as a metaphor to reflect on human restlessness and the search for purpose and satisfaction in life.
Industrial production of chemical acids glutamic acidEsam Yahya
Glutamic acid is an important amino acid that is produced industrially through fermentation using the microorganism Corynebacterium glutamicum. There are four main types of fermentation used - batch, fed-batch, continuous, and cell recycle batch fermentation. Batch fermentation is commonly used and involves inoculating a closed system with nutrients and microbes and allowing growth until nutrients are depleted. C. glutamicum is well-suited for industrial fermentation due to its rapid growth, ability to produce high yields of glutamic acid, and lack of pathogenicity. After fermentation, purification processes such as centrifugation, crystallization, and ion exchange are used to isolate glutamic acid.
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Application of a fed batch bioprocess for the heterologous production of h scomt in escherichia coli
1. J. Microbiol. Biotechnol. (2009), 19(9), 972–981
doi: 10.4014/jmb.0812.658
First published online 3 June 2009
Application of a Fed-Batch Bioprocess for the Heterologous Production of
hSCOMT in Escherichia coli
Passarinha, L. A.1*, M. J. Bonifácio2, and J. A. Queiroz1
CICS - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior 6201-001 Covilhã, Portugal
1
Departamento de Investigação e Desenvolvimento, BIAL 4745-457 São Mamede do Coronado, Portugal
2
Received: December 4, 2008 / Revised: April 8, 2009 / Accepted: April 25, 2009
In this paper, a fed-batch cultivation process in recombinant expression system is usually achieved using a two-stage
Escherichia coli BL21(DE3) bacteria, for the production of process. In the first phase, cells are grown to a high cell
human soluble catechol-O-methyltransferase (hSCOMT), density under favorable growth conditions in which protein
is presented. For the first time, a straightforward model is synthesis is kept at a minimum [22], followed by a second
applied in a recombinant hSCOMT expression system step in which high-level expression of the recombinant
and distinguishes an initial cell growth phase from a protein is achieved upon induction.
protein production phase upon induction. Specifically, the The production is affected by numerous process factors,
kinetic model predicts biomass, substrate, and product such as the cultivation mode, time of induction (with
concentrations in the culture over time and was identified respect to cell mass concentration), duration of the production
from a series of fed-batch experiments designed by testing phase, and composition of the medium [2, 29]. Many data
several feed profiles. The main advantage of this model is in literature showed that E. coli grows in salt-based
that its parameters can be identified more reliably from chemically defined media [19, 39] as long as an organic
distinct fed-batch strategies, such as glycerol pulses and carbon source is provided [22], as well as in rich complex
exponential followed by constant substrate additions. organic media. Nevertheless, higher complexities in medium
Interestingly, with the limited amount of data available, composition can lead to a lower reproducibility of the cell
the proposed model accomplishes satisfactorily the metabolic response and, therefore, lower the possibility of
experimental results obtained for the three state variables, a well-controlled process [31].
and no exhaustive process knowledge is required. The In fact, recombinant protein production processes must be
comparison of the measurement data obtained in a validation controlled by appropriately adjusting the cell environment
experiment with the model predictions showed the great [14], such as the type and concentration of macro- and
extrapolation capability of the model presented, which micronutrients. It is frequently shown that cultivation medium
could provide new complementary information for the composition directly dictates the amount of biomass
COMT production system. produced [22], and therefore can dramatically influence
Keywords: Human soluble catechol-O-methyltransferase, the desired amount of the target protein at the end of the
Escherichia coli, fed-batch bioprocess, protein production cultivation. Indeed, when E. coli is used as a host system to
produce human proteins, detailed information on acetic
acid formation should be obtained [40].
Although the improvement of the culture medium favors
Currently, recombinant human proteins as biological the attainment of high cell densities and recombinant protein
pharmaceuticals have become relevant targets in several yields, acetic acid production is also enhanced in complex
medical domains. The combination of recombinant DNA media compared with semidefined and defined media [31].
technology and large-scale culture processes has enabled Nevertheless, this main drawback in E. coli growth can be
the production of sufficient active amounts that might avoided by keeping low specific growth rates (e.g., fed-
otherwise not be obtained from natural sources [17]. The batch control algorithms on dissolved oxygen tension and
large-scale production of these proteins in the E. coli pH), by genetically altering the pathways involved in the
formation of acetate [7, 28] or merely by selecting strains
*Corresponding author
Phone: +351 275 329 069; Fax: +351 275 329 099; that have a particular genotype [27] and properly selecting
E-mail: lpassarinha@fcsaude.ubi.pt the fermentation medium components [9]. For instance,
2. RECOMBINANT HSCOMT PRODUCTION MODEL 973
recent results have demonstrated that glycerol is superior the production levels of the target protein within the
to glucose for reduced acetate and increased recombinant bacteria. A comparison of the measurement data obtained
protein production when supplied post induction [20, 26]. in the validation experiment with the model predictions
Commonly, fed-batch cultivation approaches are sufficient was performed in order to test the extrapolation capability
to achieve this scope [8]. However, much work has to be of the model. The results described are expected to provide
done in order to establish the instant and profile addition of new complementary information for this production system;
the growth-limiting carbon, often glucose [19, 32] or glycerol and regarding specific simulations, several feeding strategies
[38]. can be designed and optimized with the aim of increasing
In the past decades, recombinant soluble catechol-O- the levels of biomass for hSCOMT synthesis.
methyltransferase (SCOMT) has been produced in E. coli
[6, 25], in insect cells using a baculovirus expression system
[34], and in mammalian cell cultures using expression MATERIALS AND METHODS
vectors based on Epstein-Barr virus (a herpesvirus; [34])
and Simian virus 40 (a polyomavirus; [37]). In spite of all Chemicals
of the afore-mentioned systems having produced functional Ultrapure reagent-grade water was obtained with a Milli-Q system
(Millipore/Waters) as the basis of the bacteria growth media
forms of the enzyme, the methods enable to produce up to
formulation. Carbenicillin disodium salt, isopropylthiogalactosidase
1 g of target protein. In fact, until now, insignificant research (IPTG), tryptone, yeast extract, lysozyme, dithiothreitol, glycerol, and
has been conducted in the upstream phase for human glucose were obtained from Sigma Chemical Co. (St. Louis, MO,
SCOMT (hSCOMT) production in order to improve the U.S.A.). All other chemicals were of analytical grade commercially
volumetric and mass productivity for several biopharmaceutical available and used without further purification.
and neurological domains.
To our best knowledge, only one research group [35] Construction of the Expression Vector and Bacterial Strain
described the improvement of hSCOMT expression in The Champion pET101 Directional TOPO expression kit (Invitrogen
large scale, using a mineral modified medium with a Corporation, Carlsbad, CA, U.S.A.) was used for the expression of
substrate-limited feed strategy, by controlling the growth human SCOMT in its native form on Escherichia coli BL21(DE3)
rate with a feed-forward algorithm. The overexpression strains, and the process was carried out according to the manufacturer’s
instructions as described elsewhere [25].
makes possible to obtain a reasonable percentage of cell
protein as the desired enzyme. Nevertheless, the enzymatic Flask Experiments
activity results are not specified, and this strategy imposes Preliminary experiments were carried out in order to find the best
a significant metabolic burden on host cells. concentration and carbon source (glucose or glycerol) to be incorporated
Recently, there has been increasing awareness in the use in the developed semidefined medium (Table 1). The media under
of model predictive approaches for protein expression study had the following carbon range composition: glycerol (10, 20,
systems [10, 18]. Predictive control approaches are easy to 30, 40, and 60 g/l) and glucose (15, 20, and 30 g/l). In addition, the
understand and provide a straightforward way to explicitly pH was close to 7.2 without adjustment before sterilization, and all
handle constraints [14]. These approaches to protein process the components were mixed and dissolved in deionized water and
o
optimization and control rest on mathematical procedure autoclaved for 15 min at 120 C. After autoclaving, a specific volume
models and their exploitation with numerical optimization of a standard glucose solution was added under aseptic environment
conditions to the medium in order to obtain the desired substrate
methods. The models must be accurate enough to describe
concentration. For all flasks experiments and methods to obtain
the relevant process features, and to exploit them such that a suitable hSCOMT soluble preparation, the specific details of
the optimal values of the control variables can be determined inoculation, fermentation, and recuperation steps can be found
within the constraints imposed by the real process. elsewhere [25].
With the aim of maximizing the recombinant hSCOMT
productivity in E. coli, fed-batch cultures have been Fed-Batch Bioprocess
performed by employing several feeding strategies. This A 3.5 l bioreactor (New Brunswick Scientific, Edison, NJ, U.S.A.)
investigation hence centered on the effect of different feed containing 1.250 l of semidefined medium (Table 1) was autoclaved
o
profiles (pulses, exponential, exponential followed by at 121 C for 20 min. This medium was formulated according to
constant rate) using a substrate-limited fed-batch strategy, specific yields of main nutrients to biomass for E. coli and reported
general nutritional requirements for bacteria [41]. After sterilization,
designed to control the feed in order to maintain glycerol
magnesium sulfate heptahydratate, carbenicillin, and the trace element
below the critical level for overflow metabolism. In addition, solution (Table 1) were filter-sterilized and added separately and
we show that the relevant features of production processes aseptically to the bioreactor, in order to avoid precipitation.
for recombinant hSCOMT protein can be described by a Unless otherwise stated, the bioreactor was inoculated with
relatively simple process model. The mechanistic model 250 ml of seed culture (1.250 l starting volume), and the temperature
proposed here describes the substrate consumption by the o
was kept at 37 C throughout the fermentation with an initial aeration
E. coli cells, the accumulation in the cells’ biomass, and and agitation rate of 0.2 vvm and 250 rpm, respectively. The dissolved
3. 974 Passarinha et al.
Table 1. Medium composition for batch, fed-batch, and nutrient profiles that involved intermittent glycerol “pulses” during the growth
feed solution. phase, in order to access the influence of feed mode manipulations
Component Concentration only on the cell growth. In all experiments, we established that the
fed-batch phase started 1 h after the glycerol of the batch stage was
Na HPO
2 4 5.50 g/l exhausted.
NaCl 0.50 g/l Therefore, when the glycerol in the initial medium was depleted
Citric acid monohydrated 1.64 g/l at 9 h after inoculation, the operation mode was switched to a fed-
Potassium citrate 2.00 g/l batch with an exponential profile, where a nutrient feed (Table 1) was
Sodium citrate 0.67 g/l added initially at a flow rate of 16 ml/h (exponential rate constant
1-
Triptone 20 g/l 0.4 h ) for 4 h. When the feed rate reached a rate of 53 ml/h, it was
Glycerol 30 g/l kept constant for 7 h until the end of the cultivation. Heterologous
MgSO ·7H O 1.21 g/l hSCOMT expression was induced with the addition of 1 mM IPTG
4 2
after a predefined time. Moreover, the feed rate was adjusted to
Carbenicillin 50 µg/ml
maintain a glycerol concentration between 4 and 10 g/l during the
Trace elements 1.5 ml
production phase of the fermentation. Samples were withdrawn as
Trace elements composition necessary and stored at -20 C for analysis. o
FeCl ·6H O
3 2 27 g/l
ZnCl 2 2 g/l Process Operation
CoCl ·6H O
2 2 2 g/l Fed-batch simulations and fermentations were performed using an
Na MoO ·2H O
2 4 2 2 g/l exponential feeding profile to keep the specific growth rate constant,
CaCl ·2H O 1 g/l followed by a constant feed profile. Glycerol was the limiting substrate
2 2
CuSO 1.2 g/l and the exhaustion time was given by
4
ln ⎛ -------------- ⎞
H BO 0.5 g/l Y S
⎝ X -⎠
3 3 S ⁄X 0
Feed Composition t
0
= -------------------------- (1)
µ
lorecylg f o n o i t s u a h x e ( )
Glycerol 300 g/l xam
Triptone 40 g/l The feed solution was supplied using a peristaltic pump calibrate to
MgSO ·7H O 4 2 15 g/l impel an adequate flow specified by
Carbenicillin 250 mg/l
µ X V e xp
t t esµ ( )
Trace elements solution 10 ml/l F ( L ⁄ h) = --------------------------------------
tes 0
-0
(2)
Y S S ⁄X F
oxygen tension (DOT) monitored with an Ingold pO probe (Metter
2 Analytical Methods
Toledo) was controlled at 30% of air saturation by an agitation Cell growth determination. Dry cell weight (dcw) concentrations
cascade between 250 and 1,000 rpm and by varying the aeration rate were estimated using a calibration curve between dcw concentration
between 0.2 and 1.7 vvm. Online, pH was measured by means of an and optical density at 600 nm (OD ). The dcw values were determined 006
Ingold pH probe (Metter Toledo) and was controlled to not drop by weighing the pellet fraction from 1-ml culture samples centrifuged
o
below 7.0±0.1 by the automated addition of concentrated (2 M) at 10,000 rpm for 15 min at 4 C. The pellets were washed twice and
NaOH/NH OH or (2 M) H SO /HCl. Furthermore, foaming was
4 2 4 resuspended in 0.8% (p/v) NaCl isotonic solution, centrifuged again
o
controlled by the automated addition of an antifoam 204 agent from at the same conditions, and dried at 105 C for 48 h until constant
Sigma Chemical Co. (St. Louis, MO, U.S.A.). weight. One unit of optical density was found to be equal to 0.3862 g
3
A precultivation was carried out before each fermentation. Therefore, dcw/dm . OD was obtained from the fermentation broth, measuring
006
cells containing the expression construct were grown overnight at the absorbance at 600 nm in the range 0.2-0.9.
37 C in agar plates with standard LB medium containing 50 µg/ml
o
Glycerol, lactate, ammonium, and glucose assay. The determination
carbanecillin. A single colony was inoculated in 250 ml of a semi- of free glycerol in the supernatants of E. coli extracts (1 ml) was
defined medium (Table 1) with 50 µg/ml carbanecillin in 1 l shake based on the method previously described by Bondioli and Bella
o
flasks. The seed culture was incubated at 37 C and 250 rpm on a [5]. An YSI7100 MBS analyzer (Yellow Springs, Ohio, U.S.A.) was
rotary shaker until the optical density at 600 nm (OD ) of 2.6 was
006 employed to measure the concentration of lactate, ammonium, and
reached. Each preculture in an exponential growth phase was then glucose.
used to inoculate the bioreactor with an inoculum size of 20% (v/v). Human SCOMT activity assay. In general, the experiments of
After inoculation, the process was initially run as a batch (described activity were designed to evaluate the methylation efficiency of
in Results). Subsequently, the fed-batch strategy applied in this work recombinant hSCOMT, by measuring the amount of metanephrine,
could be projected as described elsewhere [4]: the glycerol feed using epinephrine as substrate as previously described [24]. The
ought to follow an exponential profile during the growth-phase until hSCOMT activities were calculated as nmol of MN produced/h/mg
the maximum oxygen transfer capacity is met. At this time, one protein.
should also change the glycerol profile (e.g., constant) once the
maximum stirrer speed is reached in order to avoid oxygen depletion. Model Description
Initially and in order to test the scheme depicted, after the batch One model is presented for E. coli BL21(DE3) growth intended for
phase, an exponential glycerol feed rate was compared with feed the production of recombinant hSCOMT. The model proposed is
4. RECOMBINANT HSCOMT PRODUCTION MODEL 975
unstructured and nonsegregated, which means that all cells in the The specific substrate consumption rate q is assumed to be mainly
S
population are considered to have identical properties. Identification dependent on the specific biomass growth rate µ [Eg (7)]. Additionally,
of the model structure and its parameters was based on experimental maintenance requirements of the cells and substrate uptake to protein
data obtained from fed-batch laboratory-scale cultivations. The feed production were negligible in a first approach.
strategy is designed to operate below the critical growth rate where
acetate is accumulating. Specifically, the assumptions made in the µ q P
q = ------- + ------- + m
S - - S (7)
model are as follows: Y SX Y SP
.The model does not take into consideration the complex The approached maxima q is different at different specific biomass
xam
components that are not used by the bacteria, since these growth rates. Higher specific product concentrations were observed
complex components might accumulate to inhibitive concentrations. at lower specific growth rates, and vice versa, lower specific product
. keeping out of oxygen consumption.
The concentrations were achieved at higher growth rates. In order to
. dilution rate (D) was defined, in a specific time, as the ratio
The quantify these observations, the rate of change of the specific protein
of substrate flow over bioreactor volume. concentration q in Eq. (8) was described by a first-order dynamical
p
process in the following way:
The model is based on mass balances for biomass X and substrate
S. Additionally, an equation for the specific activity of the desired q =q S I -
--------------- ------------- (8)
P xa mp
recombinant protein is considered. This balance must be distinguished S+K I+K S P
from the balances of biomass and substrate around the reactor.
This approach can be considered as a form of self-inhibition effect,
Therefore, the model was formulated in terms of p [AU/cell mass],
x since the expression of q comprises two inhibitory factors: one from
p
and does not refer to the protein concentration usually taken from
the substrate and a second from the inductor. Note that no recombinant
the lysates extracts through the induction phase. The variables
protein is produced during the biomass growth phase (I=0), and
defining the state vector c are [X, S, P ]. The following structure of
x hSCOMT production is only possible when I>0.1. Additionally, we
the mathematical model based on specific mass balances of a fed-
assume that the maximal specific product concentration value, q , xam
batch bioreactor is given by
approached asymptotically to be dependent on µ.
dX
------ = ( µ – D ) ⋅ X
- (3)
dt Parameters Estimation
A program developed in MATLAB was used for simultaneous
dS = – q ⋅ X – D ⋅ ( S – S )
-----
- S 0 (4) estimation of the model parameters, as developed by Teixeira et al.
dt
[33]. The program minimizes the mean of squared errors (MSE) using
dP - = q ⋅ X – D ⋅ P the Levenberg-Marquardt algorithm. The material balance equations
--------
X
P (5)
dt for fed-batch operation along with the postulated kinetic equations
ht ht
were integrated using a 4 /5 order Runge-Kutta solver. The estimates
For notation and values of the parameters, see Table 2.
of state variables concentration (biomass, substrate, and product) were
compared with the correspondent off-line measurements. The final
The substrate uptake rate can be described by an adaptation of the
residuals and Jacobian matrix were used to calculate an approximation
Monod kinetics relation:
to the Hessian matrix, thereby assuming that the final solution is a
--------------- ⎛ 1 – --------- ⎞
S X- local optimum. The Hessian matrix enabled the calculation of the
µ= µ (6)
K + S⎝ X ⎠
xa m
S xam parameters covariance matrix within 95% confidence intervals.
Table 2. Values of the model parameters, identified from preliminary experiments on flasks and bioreactor.
Abbreviation Value Dimensions
Operation parameters
X 0 3.7 g/l
S f 30 g/l
V 0 1.3 l
µ 0.3 h1-
Model parameters
Y S/X 0.22±0.020 biomass per glycerol (g/g)
µmáx 0.54±0.071 h 1-
a
K p 0.1 AU/l
k S 1 g/l
q x ámP 1.98±0.36 protein per biomass per h (AU/g/h)
X xá m 9.71±0.77 g/l
Y S/P 1.23±0.30 protein per glycerol (AU/g)
Reference values from Levisauskas et al. [18]
a
5. 976 Passarinha et al.
RESULTS AND DISCUSSION
Expression Conditions on Shake Flask Experiments
In order to scrutinize the best growth conditions and expression
levels of hSCOMT in the E. coli expression system applied
in this work, a shaking culture evaluation (carbon source
and concentration) was initially performed prior to the
scale up in the bioreactor. To establish the best culture
composition, the semidefined medium described in Table 1
was complemented with different compositions of a carbon
source, glycerol or glucose, through several concentrations
respectively ranging from 10 to 60 g/l for glycerol and 15,
20, and 30 g/l for glucose. These experiments were performed
in order to provide suitable information for process model
identification. Fig. 1 depicts the measurement data for biomass
concentration and hSCOMT activity levels obtained for the
two substrates at distinct concentrations.
It is well established that an excess of glucose in the Fig. 1. Comparative studies of different carbon sources (gly, glycerol;
media can lead to growth-inhibiting concentrations of acetate glu, glucose) and concentrations in terms of hSCOMT specific
activity (nmol/h/mg) and biomass (g/l) yields.
in different E. coli strains. Indeed, acetate is undesirable
since it inhibits significantly the expression of heterologous
proteins [13, 15] at considerably lower culture densities, those obtained in glucose media demonstrated that the
and represents a diversion of carbon that might otherwise reduced growth rate of E. coli observed in fermentations
generate biomass or protein product [21]. In addition, with glycerol could be more profitable, while reducing
previously conflicting results in the literature using glycerol acetate (less than 1 g/l) and maintaining hSCOMT specific
dictates that growth rate and acetate formation can be activities over all the range checked (Fig. 1). Under these
highly dependent on the recombinant strain. Therefore, to conditions, using glycerol as the exclusive carbon source
overcome these points, we decided to employ a selective at low levels in the semidefined medium, it seems that the
strain of E. coli, the BL21(DE3) Star from Invitrogen, inhibitory action of several metabolites including acetate
which possesses a particular genotype that generates less can be more straightforwardly manipulated in the bioreactor.
acetate as a result of the activation of a glyoxylate shunt Therefore, for the delineate scope, the best performance
[31, 27]. on flasks scale in terms of growth rate, biomass, and
As expected, the maximal specific hSCOMT activity volumetric hSCOMT productivity was obtained when
attained was different for the several glucose concentrations, 10 g/l of glycerol was supplement in the medium.
essentially as a result of the specific biomass growth rates
achieved and the acetate accumulation. Specifically, in the Scale-Up in a Batch Bioprocess
presence of surplus of glucose (20 and 30 g/l), the cell Subsequently, in order to examine the applicability of the
growth seemed to be inhibited, accomplished with a system described in the flasks experiments, the growth of
“proportionally” decrease in the specific activity of the E. coli cells was performed without protein induction in
expressed protein and high levels of acetate (over 4 g/l). In two ordinary batch modes with a working volume of
contrast, for a lower glucose concentration, such as 15 g/l, approximately 1.250 l. At this phase, only two glycerol
the data obtained suggest that the specific activity of the concentrations were tested (10 and 30 g/l) in order to
recombinant protein approaches to a maximal value (Fig. 1). determine the maximal specific biomass growth rate (µmax),
In spite of these analyses, this substrate concentration led the approximate yield coefficient of biomass produced as a
to acetate accumulation, which promoted several adverse function of the glycerol consumption, and the exhaustion
effects in the consequent scale-up. time of the main carbon source, to establish the beginning
Recently, several studies have demonstrated that glycerol of the fed-batch mode and consecutive profile feed. The
is superior to glucose in reducing acetate formation during choice of these glycerol concentrations was based on Fig. 1,
the growth phase, and interestingly, increased recombinant whereas the levels of hSCOMT activity obtained were
protein formation when supplied post induction [20]. In similar for the glycerol range concentration tested. In fact,
flask experiments, it was observed that an increase in the for the scale-up, testing a glycerol concentration of 30 g/l
glycerol concentration did not affect considerably hSCOMT rather than 40 g/l could be advantageous; for instance, it
production and specific activity levels. Moreover, the could decrease the acetate levels and reduce the lag phase
comparison of the results in glycerol experiments with in the bioreactor.
6. RECOMBINANT HSCOMT PRODUCTION MODEL 977
In terms of the kinetic parameters, such as specific cell After several experiments, it was established that the
growth rate, higher glycerol concentrations in the broth present feeding strategy [exponential followed by constant
medium led to lower µ (0.3 h-1), contrasting with the 0.5 h-1 feed; Fig. 2 and Eq.(8)] provided the best results in terms
estimated for 10 g glycerol/l. In both experiments, the Yxs of biomass (10 g dcw/ l in 19 h). The specific growth rate,
was found to be roughly 0.4 g cells/g glycerol, and it was µset, was fixed to 0.3 h-1 (Table 2). This value was low
also verified that E. coli stops growing when the initially enough to allow proper folding of recombinant protein, to
added amount of glycerol is consumed, 10 and 15 h avoid high oxygen transfer rates, and to keep low acetate
respectively for 10 and 30 g glycerol/l. This detail is concentrations, since the acetic acid consumption can be
enough to expand the lag phase at 30 g glycerol/l and done preferentially by the glyoxylate pathway when the
increase the batch time. This determination was confirmed glucose is not present [31].
by application of Eq. (1). As a result, when a missing Depending on the fixed production system, metabolic
substance is known in advance, it can be used as a limiting stress in E. coli is often proposed as a reason for the
factor in the biological system. Hence, in order to develop decrease in yields of a target protein during its gene
a suitable fed-batch strategy, it was established that the expression, which can result in the redirection of cellular
batch phase should be performed at 10 g/l of glycerol metabolism [12] and a drop off in the growth rate [1].
during 9 h, followed by a feed profile strategy. These effects have been reported in some biological systems,
essentially due to the addition of the inducer IPTG [3, 16].
Production of hSCOMT by a Fed-Batch Bioprocess Once growth conditions and criteria for inductor quantity
In a first approach, the yield coefficient (YX/S) was estimated have been fixed, the induction time is the manipulated
from the fed-batch experiments through the use of glycerol variable to maximize production yields.
balance measurements. In fact, this parameter is close to In this work, we circumvented the need to determine the
the estimation done during the batch experiment: around effect of the induction time on cell growth and recombinant
0.35 and 0.5 g cells/g glycerol, respectively, for exponential protein yields, since as described previously [35], the final
and pulse feeding modes. These experiments also showed yield of SCOMT was not dependent on the culture density
that supplementation of glycerol by an intermittent feed at the moment of the induction. Hence, although the
mode operation (three additions) was an inadequate strategy, concentration of the produced protein was to a great extent
since unusually, the glycerol concentration was constant at proportional to cell concentration, and processes designed
the predefined set-point (10 g/l). The results reveal that a good to yield high cell densities are beneficial, we decided to
alternative was to follow an exponential glycerol profile
during the exponential stage, to restore gradually and overtime
the glycerol contents in the bioreactor around 10 g/l.
From bibliographical data, one way to prevent oxygen
depletion is to lower the fermentation temperature after
induction, since low efficiency in active recombinant protein
production can occur when maintaining the temperature of
37oC, for example, because of inclusion bodies formation
related to high growth rate during recombinant expression
[11, 30]. In this work, the majority of the target protein was
found in an active and soluble form, and no reduction of
temperature was necessary because the growth rate was
partially controlled during the fed-batch phase owing to
the imposition of lower glycerol levels.
Therefore, to maximize recombinant hSCOMT production
by achieving a maximal final culture density (g dcw/dm3)
and also maximal specific activity (U/g dcw) at the end of
the growth, we decided to establish an exponential profile
during the growth phase followed by a constant feed over
the induction stage. In this phase, the maximum oxygen
transfer capacity of the system is attained, and the biomass
levels could decrease mainly because of oxygen limitations.
Consequently, a control between agitation and oxygen Fig. 2. The behavior of cultivation parameters during fed-batch
dissolved levels had to be done in order to ensure a maximum procedure of hSCOMT expression. The starting of the feeding and
induction are indicated by the left and right arrow, respectively.
stirrer speed in the medium without possible oxygen OD, optical density [dry cell weight (g/l)=0.3863 s OD]; X,
depletions. biomass concentration (g/l).
7. 978 Passarinha et al.
perform the recombinant protein induction by one pulse
IPTG addition at a dry cell weight of 8.8 g/l at the late
feeding phase (Fig. 2).
It is well known that a high-energy demand for a
nongrowth-related process (in this case, the synthesis of
the recombinant protein) can decrease the observed biomass
yield coefficient [23, 36]. This behavior was observed in
the fed-batch tested (Fig. 2), and in spite that the culture
reached automatically in the stationary phase, the volumetric
hSCOMT accumulation increased significantly from 92,187
to 583,975 (U/l) in three hours of induction (Fig. 3);
approximately 41% higher than in a routine flask experiment
for the same experimental conditions. Another fact is that
we could prolong the induction time (e.g., for three more
hours), since the reduction of biomass concentration with
the associated pseudo inhibitory effects of the induction
stage were not dramatically observed as in the literature for
other recombinant proteins in high cell density systems.
Model Design
A model was designed based on Monod kinetics that
incorporates biomass inhibition, and substrate and inductor
limitation with a straightforward approach for protein
production, which was used to predict biomass, glycerol,
and hSCOMT levels over the fermentation run. In Fig. 4, a
simulation of the model of Escherichia coli BL21(DE3)
with an exponential/constant (4A) and a pulse (4B) glycerol Fig. 3. Chromatograms obtained from the incubation medium for
feed is demonstrated. In a first approach, we decided to recombinant hSCOMT activity assay: (A) 1 h of induction in a
neglect the lag phase data in terms of biomass and substrate bioreactor and (B) 3 h of induction in a bioreactor.
concentrations in order to improve the results between
experimental values and model approach. The curves for between prediction and experimental data was observed,
biomass and the substrate depict the corresponding modeling indicating the high-quality prediction properties of this
results obtained after fitting the model to all three data sets straightforward model. Furthermore, the comparison of
with the same parameter set (see Table 2). hSCOMT yields in shake flasks with this fed-batch
The resulting fits show that the agreement among model bioreactor strategy led to a significant improvement in the
and measured data is satisfactory, particularly if one takes process performance. Fig. 5 also shows that a maximum of
into account the significantly different operational feeding specific activity can be reached after 3 h of induction;
conditions under which the process was examined. In however, this final concentration could be increased if
particular, the abrupt increase in glycerol concentration induction were prolonged four hours more in a higher
shown in the model (Fig. 4A) was not confirmed by the biomass concentration. Nevertheless, specific protein activities
experimental data, probably owing to an initial accumulation in the cell mass were found in the predicted range, and the
of the substrate at the earlier growth phase. However, after total productivity of the process also met the predicted
the protein induction instance the glycerol concentration values.
dropped off quickly, since the growth and metabolic
activity of the host cells were influenced by the expression Concluding Remarks
of the heterologous protein [39]. For the experiment In this work, a straightforward knowledge of (a) the maximum
described in Fig. 4B (intermittent pulses of glycerol), our allowable biomass concentration in the fermentation
measurement data were satisfactorily accurate when compared system, (b) the growth and protein evolution behavior after
with the representation proposed by the developed model. induction, and (c) the inductor concentration assayed by
the final expected biomass, show how to apply an exponential
Validation Experiments followed by a constant fed-batch strategy to get satisfactory
Finally, analysis of the process performance was tested in a yields in recombinant hSCOMT expression systems. The
validation experiment accomplished with the production of mathematical model proposed for simple parameters prediction
recombinant hSCOMT protein (Fig. 5). A good agreement and further optimization of the operational procedure in
8. RECOMBINANT HSCOMT PRODUCTION MODEL 979
Fig. 4. Biomass levels and glycerol concentrations during experimental bioreactor trials using (A) exponential followed by constant
glycerol fed-batch operation mode and (B) intermittent glycerol profiles.
The plots were accomplished with results predicted by simulations using the model proposed for biomass and substrate concentration.
recombinant hSCOMT production processes reliably YPS: product/substrate yield, g/g
predict the experimental data. The above methodology was X: biomass amount in fermenter, g
used in the determination of the proper conditions for high- Xmáx : maximal biomass amount in fermenter, g;
level hSCOMT production, achieving a final volumetric SF: substrate concentration in feeding solution g/l
accumulation after 3 h of induction around 581,780 U/l. S: substrate concentration, g/l
The results described are expected to provide new S0: substrate concentration in the feed, g/l
complementary information for this specific production qS: specific substrate consumption rate, g/g/h
system, and regarding these simulations, a feeding strategy qP: specific protein accumulation rate, AU/g/h
can be designed and optimized with the aim of reducing qPmax: maximal specific protein accumulation, AU/g/h
the levels of substrate in order to increase the levels of KS: Monod constant for substrate consumption, g/l
biomass and hSCOMT protein. D: Dilution rate, 1/h
I: Inductor concentration; g/l
List of Symbols
Acknowledgment
µ: specific biomass growth, 1/h
µmáx: maximal specific biomass growth, 1/h The kind collaboration of João Dias from Faculdade de
YXS: biomass/substrate yield, g/g Ciências e Tecnologia, Universidade Nova de Lisboa, by
9. 980 Passarinha et al.
Fig. 5. Validation profiles of the process variables in a fed-batch experiment (exponential followed by constant profile) with the
production of the recombinant hSCOMT protein.
providing us material and technical support for the 5. Bondioli, P. and L. D. Bella. 2005. An alternative spectrophotometric
development of this work, is gratefully acknowledged. method for the determination of free glycerol in biodiesel. Eur.
J. Lipid Sci. Technol. 107: 153-157.
6. Bonifácio, M. J., M. A. Vieira-Coelho, and P. Soares-da-Silva.
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