Browsing by Author "Gonzalez, Ramon"

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    A Novel Human Adipocyte-Derived Basement Membrane for Tissue Engineering Applications
    (2012-09-05) Damm, Aaron; Nagrath, Deepak; Zygourakis, Kyriacos; Gonzalez, Ramon; Sabek, Omaima
    Tissue engineering strategies have traditionally focused on the use of synthetic polymers as support scaffolds for cell growth. Recently, strategies have shifted towards a natural biologically derived scaffold, with the main focus on decellularized organs. Here, we report the development and engineering of a scaffold naturally secreted by human preadipocytes during differentiation. During this differentiation process, the preadipocytes remodel the extracellular matrix by releasing new extracellular proteins. Finally, we investigated the viability of the new basement membrane as a scaffold for tissue engineering using human pancreatic islets, and as a scaffold for soft tissue repair. After identifying the original scaffold material, we sought to improve the yield of material, treating the cell as a bioreactor, through various nutritional and cytokine stimuli. The results suggest that adipocytes can be used as bioreactors to produce a designer-specified engineered human extracellular matrix scaffold for specific tissue engineering applications.
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    Anaerobic fermentation of glycerol
    (2012-03-06) Gonzalez, Ramon; Rice University; United States Patent and Trademark Office
    The invention relates to the development of appropriate cultivation conditions for a bacteria to grow anaerobically (fermentatively) on a glycerol substrate. The method requires culturing bacteria having a functional 1,2-propanediol pathway and a functional type II glycerol dehydrogenase-dihydroxyacetone kinase pathway in a culture medium containing high concentrations of glycerol, a neutral to mildly acidic pH, low levels of potassium and phosphate, and high levels of CO2, such that glycerol is thus converted into a desirable product, such as ethanol, hydrogen, formate, succinate, or 1,2-propanediol.
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    Anaerobic fermentation of glycerol
    (2012-12-18) Gonzalez, Ramon; Rice University; United States Patent and Trademark Office
    The invention relates to the development of appropriate cultivation conditions for a bacteria to grow anaerobically (fermentatively) on a glycerol substrate. The method requires culturing bacteria having a functional 1,2-propanediol pathway and a functional type II glycerol dehydrogenase-dihydroxyacetone kinase pathway in a culture medium containing high concentrations of glycerol, a neutral to mildly acidic pII, low levels of potassium and phosphate, and high levels of CO2, such that glycerol is thus converted into a desirable product, such as ethanol, hydrogen, formate, succinate, or 1,2-propanediol.
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    Anaerobic fermentation of glycerol: a platform for renewable fuels and chemicals
    (Cell Press, 2013-01) Clomburg, James M.; Gonzalez, Ramon; Bioengineering; Chemical and Biomolecular Engineering
    To ensure the long-term viability of biorefineries, it is essential to go beyond the carbohydrate-based platform and develop complementing technologies capable of producing fuels and chemicals from a wide array of available materials. Glycerol, a readily available and inexpensive compound, is generated during biodiesel, oleochemical, and bioethanol production processes, making its conversion into value-added products of great interest. The high degree of reduction of carbon atoms in glycerol confers the ability to produce fuels and reduced chemicals at higher yields when compared to the use of carbohydrates. This review focuses on current engineering efforts as well as the challenges involved in the utilization of glycerol as a carbon source for the production of fuels and chemicals.
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    Antibiotic resistance vector transport, reservoir amplification and attenuation
    (2008) Rysz, Michal; Alvarez, Pedro J.; Ward, C. H.; Gonzalez, Ramon
    Microbial antibiotic resistance is an emerging environmental pollution problem with deleterious effects on water supplies and human health. Currently, little is known about the role of environmental factors in the maintenance, propagation and attenuation of antibiotic resistance. This study investigated the effects of antibiotic exposure concentrations, nutrient availability, and microbial growth rate on resistance dynamics, as well as, the porous medium transport characteristics of antibiotic resistance vectors. Exposure to high antibiotic concentrations increased, (1) the percentage of resistant bacterial strains in soil, (2) the persistence of resistant strains in soil and (3) the relative abundance of resistance genes in bacteria; and decreased the Shannon Weaver diversity index. Rich growth medium enhanced resistance plasmid maintenance and stability even in the absence of selective pressure of the antibiotic possibly be alleviating the metabolic burden imparted on the carrier bacteria by the resistance plasmids. The growth rate exerted a strain-specific response on resistance dynamics, with higher plasmid instability (i.e., increased loss) observed at higher growth rates (Pseudomonas aeruginosa), but no such effect observed for an Eschericha coli strain. Resistance vector plumes may be enhanced by: (1) groundwater conditions conducive to plasmid coagulation and colloid formation of approximately 1 mum, and (2) high concentrations of resistant bacteria that exhibit fast initial deposition, and strong blocking behavior after matrix deposition. The results of this research suggest that decreasing environmental antimicrobial concentrations will be conducive to the attenuation of microbial antibiotic resistance, but may not be sufficient in completely eliminating the resistance reservoirs, thus additional control methods may be needed to minimize the impact of these pollutants. The results should also provide insight to improve regulatory and sustainability decision-making processes related to the use of antibiotic in animal agriculture.
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    Catalytic Oxidation Properties of Palladium-decorated Gold Nanoparticles
    (2014-10-06) Zhao, Zhun; Wong, Michael S.; Gonzalez, Ramon; Zheng, Junrong
    Bimetallic palladium gold (PdAu) catalysts have been shown to be superior to monometallic ones in many reactions, but the reasons for the enhancement are not thoroughly understood. In this work, palladium decorated gold nanoparticles (Pd-on-Au NPs) are used as structured model catalysts, allowing for the precise control of both size and metal distribution with Pd surface coverage (sc%). By testing reactions on a range of these catalysts, we hope to gain insight into the active site for a given reaction. In hydrodechlorination of perchloroethene (PCE), Pd surface coverage was found to be the key factor in catalyst activity, with the optimum at 80 sc%. A complete mechanistic model that coupled mass transfer processes with the surface reactions was further developed, consistent with the observed product profiles. Carbon supported Pd-on-Au NPs were tested for liquid phase glycerol oxidation for the first time. The best catalyst (80 sc%) had an initial TOF of ~6000 h-1, >10 times more active than Au/C and Pd/C. Catalytic activity, selectivity, activation energy and deactivation rate constant exhibited strong volcano-shaped dependences upon Pd sc%. Ex situ XANES results showed no to little change in surface Pd-O% for Au based catalysts, suggesting the possibility of Au suppressing Pd oxidation during reaction. Ex situ EXAFS results further confirmed the core-shell structures of 60 and 150 sc% Pd-on-Au/C catalysts via Punnett square analysis, and also ascertained no to little change in their oxidation states and coordination numbers post glycerol oxidation. EXAFS observations correlate with kinetics results, and lead to the conclusion that catalysts with a larger amount of 3-D Pd ensembles are more prone to oxidize during glycerol oxidation, making them less resistant to deactivation. Finally, Pd-on-Au/C catalysts were tested for room temperature formic acid decomposition. In situ XAS revealed that core-shell structures of 60, 150 and 300 sc% Pd-on-Au NPs maintained while oxidized Pd species was partially reduced during reaction. Catalyst with higher fraction of 3-D Pd ensembles showed much higher dehydrogenation activity than those with mostly 1-D or 2-D, correlating to the proposed mechanism that the dehydrogenation pathway is favored over metal terrace sites.
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    Development of de novo biosynthetic pathways
    (2017-08-10) Cheong, Seokjung; Gonzalez, Ramon
    Microbial biorefineries and biomanufacturing, which harness biosynthetic pathways, are becoming economically viable alternatives to traditional petrochemical refineries and manufacturing due to their cleanness, flexibility, and usage of biorenewable or recycled feedstocks. However, the energy or carbon efficiencies and product diversity of biosynthetic pathways are not sufficient for scaled-up industrial processes. The goal of this thesis is to develop de novo biosynthetic pathways with improved energy or carbon efficiencies and/or product diversity as alternatives to three widely used biosynthetic pathways: β-oxidation reversal, isoprenoid biosynthesis and polyketide biosynthesis. A novel, iterative, modular, combinatorial and orthogonal carbon-carbon elongation platform composed of non-decarboxylative Claisen condensation reactions and susbsequent β-reductions was developed. This platform follows a similar mechanism and exhibits equivalent high carbon and energy efficiencies to those of the β-oxidation reversal, but in addition is able to accept various ω-, and ω-1-functionalized primers and α-functionalized extender units and synthesize diverse chemicals with α-, β-, ω-, and ω-1-functionalities. During the study, the production of 18 compounds from 10 product classes was demonstrated with noticeable titers. Among them, seven compounds were produced for the first time via microbial fermentations. This thesis also proposed four novel pathways for the biosynthesis of isoprenoid building blocks dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), which are more energy- or carbon-efficient than native mevalonate or non-mevalonate pathways. Though the search of some enzymes required for realizations of these pathways are still underway, many enzymatic components have been characterized in vitro and in vivo and utilization of confirmed components led to efficient microbial productions of 2-hydroxyisovalerate and prenol with titers of 8.46 g/L and 0.48 g/L respectively. A novel, energy-efficient polyketide biosynthesis pathways has also been designed as an alternative to native, polyketide synthase (PKS)-based routes. Instead of using PKSs, the pathway is based on repetitive non-decarboxylative Claisen condensation reactions catalyzed by thiolases, which bypasses the ATP-consuming carboxylation step required for the generation of extender units used by PKSs. Among thiolases tested for polyketide synthesis, BktB, ScFadA, FadAx and DcaF showed promising signs of production of pyrone triacetic acid lactone (TAL) through condensation between acetoacetyl-CoA and acetyl-CoA with highest titer as 0.36 g/L. FadAx also promisingly exhibited in vitro condensation activity between two acetoacetyl-CoAs to generate another pyrone dehydroacetic acid. Achievements of this thesis, in line with subsequent improvements, are expected to highly benefit the development of microbial biomanufacturing processes and biorefineries.
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    Differential Effect of Culture Temperature and Specific Growth Rate on CHO Cell Behavior in Chemostat Culture
    (Public Library of Science, 2014) Vergara, Mauricio; Becerra, Silvana; Berrios, Julio; Osses, Nelson; Reyes, Juan; Rodriguez-Moya, Maria; Gonzalez, Ramon; Altamirano, Claudia; Bioengineering; Chemical and Biomolecular Engineering
    Mild hypothermia condition in mammalian cell culture technology has been one of the main focuses of research for the development of breeding strategies to maximize productivity of these production systems. Despite the large number of studies that show positive effects of mild hypothermia on specific productivity of r-proteins, no experimental approach has addressed the indirect effect of lower temperatures on specific cell growth rate, nor how this condition possibly affects less specific productivity of r-proteins. To separately analyze the effects of mild hypothermia and specific growth rate on CHO cell metabolism and recombinant human tissue plasminogen activator productivity as a model system, high dilution rate (0.017 h21) and low dilution rate (0.012 h21) at two cultivation temperatures (37 and 33ﾰC) were evaluated using chemostat culture. The results showed a positive effect on the specific productivity of r-protein with decreasing specific growth rate at 33ﾰC. Differential effect was achieved by mild hypothermia on the specific productivity of r-protein, contrary to the evidence reported in batch culture. Interestingly, reduction of metabolism could not be associated with a decrease in culture temperature, but rather with a decrease in specific growth rate.
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    Efficient synthesis of L-lactic acid from glycerol by metabolically engineered Escherichia coli
    (BioMed Central, 2013) Mazumdar, Suman; Blankschien, Matthew D.; Clomburg, James M.; Gonzalez, Ramon; Bioengineering; Chemical and Biomolecular Engineering
    Due to its abundance and low-price, glycerol has become an attractive carbon source for the industrial production of value-added fuels and chemicals. This work reports the engineering of E. coli for the efficient conversion of glycerol into L-lactic acid(L-lactate). Escherichia coli strains have previously been metabolically engineered for the microaerobic production of D-lactic acid from glycerol in defined media by disrupting genes that minimize the synthesis of succinate, acetate, and ethanol, and also overexpressing the respiratory route of glycerol dissimilation (GlpK/GlpD). Here, further rounds of rationale design were performed on these strains for the homofermentative production of L-lactate, not normally produced in E. coli. Specifically, L-lactate production was enabled by: 1), replacing the native D-lactate specific dehydrogenase with Streptococcus bovis L-lactate dehydrogenase (L-LDH), 2) blocking the methylglyoxal bypass pathways to avoid the synthesis of a racemic mixture of D- and L-lactate and prevent the accumulation of toxic intermediate, methylglyoxal, and 3) the native aerobic L-lactate dehydrogenase was blocked to prevent the undesired utilization of L-lactate. The engineered strain produced 50 g/L of L-lactate from 56 g/L of crude glycerol at a yield 93% of the theoretical maximum and with high optical (99.9%) and chemical (97%) purity. This study demonstrates the efficient conversion of glycerol to L-lactate, a microbial process that had not been reported in the literature prior to our work. The engineered biocatalysts produced L-lactate from crude glycerol in defined minimal salts medium at high chemical and optical purity.
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    Endoplasmic Reticulum-Associated rht-PA Processing in CHO Cells: Influence of Mild Hypothermia and Specific Growth Rates in Batch and Chemostat Cultures
    (Public Library of Science, 2015) Vergara, Mauricio; Berrios, Julio; Martínez, Irene; Díaz-Barrera, Alvaro; Acevedo, Cristian; Reyes, Juan G.; Gonzalez, Ramon; Altamirano, Claudia; Bioengineering; Chemical and Biomolecular Engineering
    Background: Chinese hamster ovary (CHO) cells are the main host for producing recombinant proteins with human therapeutic applications mainly because of their capability to perform proper folding and glycosylation processes. In addition, mild hypothermia is one of the main strategies for maximising the productivity of these systems. However, little information is available on the effect of culture temperature on the folding and degradation processes of recombinant proteins that takes place in the endoplasmic reticulum. Methods: In order to evaluate the effect of the mild hypothermia on processing/endoplasmatic reticulum-associated degradation (ERAD) processes, batch cultures of CHO cells producing recombinant human tissue plasminogen activator (rht-PA) were carried out at two temperatures (37°C and 33°C) and treated with specific inhibitors of glycosylation and ERAD I (Ubiquitin/Proteasome system) or ERAD II (Autophagosoma/Lisosomal system) pathways. The effect of mild hypothermia was analysed separately from its indirect effect on specific cell growth rate. To do this, chemostat cultures were carried out at the same incubation conditions as the batch cultures, controlling cell growth at high (0.017 h-1) and low (0.012 h-1) dilution rates. For a better understanding of the investigated phenomenon, cell behaviour was also analysed using principal component analysis (PCA). Results and Conclusion: Results suggest that rht-PA is susceptible to degradation by both ERAD pathways studied, revealing that processing and/or ERAD processes are sensitive to temperature cultivation in batch culture. Moreover, by isolating the effect of culture temperature from the effect of cell growth rate verifyed by using chemostat cultures, we have found that processing and/or ERAD processes are more sensitive to reduction in specific growth rate than low temperature, and that temperature reduction may have a positive effect on protein processing. Interestingly, PCA indicated that the integrated performance displayed by CHO cells is modulated predominantly by specific growth rate, indicating that the culture temperature has a lower weighted effect within the range of conditions evaluated in this work.
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    Engineering Escherichia coli for the production of polyketide-based platform chemicals
    (2012) Park, John; Gonzalez, Ramon
    The current chemical industry produces a diverse array of industrial chemicals from a handful of highly reduced byproducts (termed "platform chemicals") derived from oil refining. However, petroleum is a non-renewable resource, and increases in its cost have created pressure to convert the chemical industry into one that is renewable to ensure its long-term viability. To complete this objective, one approach is the conversion of biomass to platform chemicals through fermentation by Escherichia coli . One such platform chemical is methyl ketone, which can be readily converted to dienes that can directly replace existing platform chemicals such as ethylene. To bestow non-native methyl ketone production capability to E. coli from glucose, the polyketide biosynthesis pathway was exploited in conjunction with grafting in a heterologous methyl ketone synthesis pathway found in wild tomato species Solanum habrochaites to produce the methyl ketones. Cultivation under microaerobic conditions improved titers and yields, and further engineering to knock out the native competitive pathways that become activated under microaerobic conditions led to significantly improved strains. The final strain, ΔadheΔldhaΔptaΔpoxB [pTrcHis2A-shmks2-mks1], produced up to 450 mg/L of methyl ketones at 17 mg of methyl ketones produced per gram of glucose consumed under optimized operating conditions in minimal media supplemented with glucose.
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    Engineering of microbial cell factories for the sustainable production of fuels and chemicals using a novel carbon elongation pathway
    (2017-08-10) Kim, Seohyoung; Gonzalez, Ramon
    Concerns over sustained availability of fossil resources along with environmental impact of their use have stimulated the development of alternative methods for fuel and chemical production from renewable resources. Feedstocks from renewables are commonly C5~C6 sugars and C3 glycerol obtained from hydrolytic degradation of biomass and lipids respectively, and some successful results in production of chemicals in the C1-C6 range have been reported largely due to fact that those chemicals can be produced directly or be readily synthesized from common compounds within central metabolism. The biomanufacturing of chemicals of which the number of carbon exceed 6, on the other hand, requires elaborate intracellular carbon-carbon bond forming reactions. Although numerous studies have employed nature designed carbon elongation pathway such as fatty acid biosynthesis and isoprenoid pathway in attempt to produce those chemicals, most metabolic platforms suffer from low titer and yield of products largely because of both cellular regulation and high expenditure of cellular energy on the biosynthesis of starter and extender units for carbon elongation. These obstacles motivated us to an investigation of new approaches to develop cell factories to enable sustainable production of chemicals. To this end, we selected the reversed β-oxidation (r-BOX) as carbon elongation module due to its orthogonality and superiority in energy consumption and functionality compared with that of other carbon elongation modules. The construction of a functional r-BOX, however, was only achieved by manipulating multiple global regulators which rendered it less applicable to regulate carbon elongation and fine-tuning the addition of functional groups. To address these difficulties, we adopted a bottom-up synthetic biology approach to facilitate r-BOX in E. coli which consist of four individual enzymatic reactions: thiolase, 3-hydroxy acyl-CoA dehydrogenase, trans-enoyl-CoA dehydratase and 2-enoyl-CoA reductase. In addition, further assessment on the efficiency of newly developed cell factories was carried out based on the three criteria: i) carbon elongation where the biosynthesis of decanoyl-CoA from acetyl-CoA was confirmed by using corresponding n-acids and n-alcohols as proxy products, ii) functionality on the carbon back bone where the production of α,β-unsaturated carboxylic acids were demonstrated and iii) selectivity in carbon chain length using decanoic acid as a proxy product. In these respects, the cell factories implemented with r-BOX along with various acyl-CoA hydrolysis module exhibited superiority in titer, yield and ratio. Thus, we demonstrated that metabolic engineering strategies covered in the present study to develop cell-factories were useful as a tool for the production of various chain length of chemicals with both different functional groups and high yield.
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    Fermentative utilization of glycerol and lignocellulosic sugars and production of ethanol by Paenibacillus macerans
    (2010) Gupta, Ashutosh; Gonzalez, Ramon
    With the recent volatility in crude oil prices and widespread concern regarding global warming, there is increased need for finding sustainable alternatives to petrochemical based fuels. One way to achieve this objective is through the production of biofuels such as ethanol by microbial fermentation. In this context, this study focuses on the anaerobic fermentation of renewable substrates; glycerol and lignocellulosic sugar mixtures for the production of ethanol by P. macerans. Further, metabolic flux analysis (MFA) was used as a systemwide tool to understand the role of various metabolic pathways in the fermentative utilization of these substrates. Glycerol is a byproduct of biodiesel and bioethanol production, which is an abundant, inexpensive and renewable substrate. In this study, we have shown that P. macerans can anaerobically ferment glycerol in the absence of external electron acceptors. Nuclear magnetic resonance (NMR) analysis of the fermentation samples identified the production of ethanol, formate, acetate, succinate, and 1,2-propanediol (1,2-PDO) from glycerol. Use of U-13C glycerol as substrate demonstrated the incorporation of glycerol in the cell biomass. Glycerol fermentation was stimulated in a medium formulation with low concentrations of potassium and phosphate, cultivation at acidic pH, and the use of a CO2-enriched atmosphere. Since the consumption of reducing equivalents in the production of 1,2-PDO balances the reducing equivalents generated in the production of cell biomass, the synthesis of ethanol and 1,2-PDO are proposed to be a metabolic determinant of glycerol fermentation in P. macerans. Hexose and pentose sugars make the largest portion of lignocellulosic biomass, which is the ideal substrate for the production of biofuels. Most microorganisms can not utilize hexose and pentose sugars simultaneously. In this study, we have shown that P. macerans N234A can ferment hexose (glucose) and pentose (xylose and arabinose) sugars individually in the absence of external electron acceptors. Additionally, we have shown that P. macerans N234A can simultaneously utilize the three sugars of lignocellulosic biomass. We have also identified the factors pH and temperature, which improve the simultaneous sugar utilization by this microorganism. Metabolic flux analysis was used as an in vivo tool for systemwide study of glycerol and sugar mixture fermentations to elucidate the role of various pathways. In the case of glycerol fermentation, MFA analysis identified the role of 1,2-PDO production in achieving the redox balance by consuming the redox equivalents being generated in the production of biomass. Flux analysis also showed the role of PFL in pyruvate dissimilation in the glycerol fermentation. Similarly, in the case of sugar mixture, MFA analysis showed the role of PFL and PDH enzymes in the utilization of sugar mixtures by P. macerans.
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    Functionalized carboxylic acids and alcohols by reverse fatty acid oxidation in engineered microbes
    (2018-06-12) Gonzalez, Ramon; Clomburg, James M.; Rice University; United States Patent and Trademark Office
    Bacteria that run the beta oxidation cycle in reverse anabolic direction are provided, along with many novel primers to start the reverse cycle, pathways to make such primers, and a large variety of products produced thereby. Methods for making desired product by using such primers in the reverse pathway are also disclosed.
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    Improved Understanding of Apoptosis and Metabolism in Chinese Hamster Ovary Cell Culture
    (2011) Sun, Ruiqiang; Gonzalez, Ramon
    Mammalian cell culture has gained importance in biotechnology for the development of therapeutic and diagnostic agents. Among them, Chinese hamster ovary (CHO) cells are regarded as the mammalian cell "workhorse". The use of CHO cell line for the production of recombinant proteins used in human therapy has reached a level of industrial production. However, a major problem encountered in in vitro cultures is cell death via apoptosis. Since apoptosis leads to the loss of viability of mammalian cells in vitro, especially in serum-free media. This is important and necessary to prevent the activation of apoptosis cascade and increase their cell viability and enhance their cellular robustness. The overall goal of this study is to improve our understanding of the cellular and physiological determinants of apoptosis and its relationship with other cellular functions. Apoptosis is a result of a very complex network of signaling pathways triggered from both inside and outside of the cell and a highly regulated pathway by both pro-apoptotic and anti-apoptotic proteins that promote cell survival or cell death. Although many causes of apoptotic process in mammalian cell cultures had been researched in the past and have been discussed in recent years, a lot need to be explored. In order to bring novel strategies to understand apoptosis in mammalian cell cultures, our study was not only focused on the apoptotic pathway but also expand to metabolic network to set up a link between cell growth and apoptosis. In our project, we applied systems biology methods in a mammalian cell line (CHO TF 70R), to understand the relationship between cellular metabolism and apoptosis in a typical serum free culture medium. After establishing the basic culture platform, the effects of culture conditions on initiating apoptosis will be evaluated. Healthy and apoptotic cell samples were identified and isolated using Fluorescence Activated Cell Sorting (FACS) and Magnetic Activated Cell Sorting (MACS), respectively. A comprehensive study of CHO cellular metabolism was made using a metabolic flux network to compare and analyze by metabolic flux analysis (MFA) to get more information on cell metabolism and apoptotic behavior. Furthermore, 2-NBDG combined with Annexin V-PE was also successfully applied to estimate the glucose uptake rate in real early apoptotic cells. In summary, we used the integration of the data generated by MFA to understand apoptotic behavior and establish a correlation between cell regulation and apoptosis. It will help us to identify the changes during the onset of apoptosis process will be studied by using proteomics tools to analyze the protein up-regulation or down-regulation in different cell status in the future.
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    In silico and in vivo analyses reveal key metabolic pathways enabling the fermentative utilization of glycerol in Escherichia coli
    (Wiley, 2022) Clomburg, James M.; Cintolesi, Angela; Gonzalez, Ramon
    Most microorganisms can metabolize glycerol when external electron acceptors are available (i.e. under respiratory conditions). However, few can do so under fermentative conditions owing to the unique redox constraints imposed by the high degree of reduction of glycerol. Here, we utilize in silico analysis combined with in vivo genetic and biochemical approaches to investigate the fermentative metabolism of glycerol in Escherichia coli. We found that E. coli can achieve redox balance at alkaline pH by reducing protons to H2, complementing the previously reported role of 1,2-propanediol synthesis under acidic conditions. In this new redox balancing mode, H2 evolution is coupled to a respiratory glycerol dissimilation pathway composed of glycerol kinase (GK) and glycerol-3-phosphate (G3P) dehydrogenase (G3PDH). GK activates glycerol to G3P, which is further oxidized by G3PDH to generate reduced quinones that drive hydrogenase-dependent H2 evolution. Despite the importance of the GK-G3PDH route under alkaline conditions, we found that the NADH-generating glycerol dissimilation pathway via glycerol dehydrogenase (GldA) and phosphoenolpyruvate (PEP)-dependent dihydroxyacetone kinase (DHAK) was essential under both alkaline and acidic conditions. We assessed system-wide metabolic impacts of the constraints imposed by the PEP dependency of the GldA-DHAK route. This included the identification of enzymes and pathways that were not previously known to be involved in glycerol metabolisms such as PEP carboxykinase, PEP synthetase, multiple fructose-1,6-bisphosphatases and the fructose phosphate bypass.
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    Kinetic and statistical approaches in metabolic networks
    (2008) Rigou, Venetia; Gonzalez, Ramon
    The work in the current thesis is focused on the metabolomics field. In general, studies in this area concentrate on obtaining information concerning the metabolites found in a biological sample. Acquiring and analyzing metabolome data provides a better understanding of the bionetwork's mechanisms. The first part of the thesis focalizes on analyzing metabolome data measured from plant systems. The proposed methodology is based on Principal Component Analysis. The primary goal is to reduce the dimensionality of a large dataset while retaining most of the associated information. The aforementioned methodology additionally provides the chance to obtain assay signatures by using metabolite contributions. Its importance relies on the fact that we are able to define the biomarkers in a biological sample and thus, providing a mean to further examine a desirable cellular function. The second part of the thesis introduces the development of a kinetic model, implemented to describe the anaerobic fermentation of glycerol in Escherichia coli. Setting up the overall model required obtaining the parameters from the literature, and determining the kinetic expressions for each enzyme. The results include the acquisition of the metabolite profiles over time, plus their steady-state concentrations. Additionally, Metabolic Control Analysis was applied to the steady-state conditions, in order to obtain an insight of which enzymes can be considered responsible for the control of flux. The accuracy of the outcome could be verified if the computational efforts were combined with experimental work. The results derived from the kinetic modeling of the anaerobic fermentation of glycerol underline the importance of utilizing methodologies that eliminate the need of kinetic data, like metabolic flux analysis, or the incorporation of non-mechanistic methods, like the log-linear approximation.
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    Kinetic and Stoichiometric Modeling of the Metabolism of Escherichia coli for the Synthesis of Biofuels and Chemicals
    (2013-09-16) Cintolesi Makuc, Angela; Gonzalez, Ramon; Nagrath, Deepak; Nakhleh, Luay K.
    This thesis presents the mathematical modeling of two new Escherichia coli platforms with economical potential for the production of biofuels and chemicals, namely glycerol fermentation and the reversal of the β-oxidation cycle. With the increase in traditional fuel prices, alternative renewable energy sources are needed, and the efficient production of biofuels becomes imperative. So far studies have focused on using glucose as feedstock for the production of ethanol and other fuels, but a recent increase in glycerol availability and its consequent decrease in price make it an attractive feedstock. Furthermore, the reversed β-oxidation cycle is a highly efficient mechanism for the synthesis of long-chain products. These two platforms have been reported experimentally in E. coli but their mathematical modeling is presented for the first time here. Because mathematical models have proved to be useful in the optimization of microbial metabolism, two complementary models were used in this study: kinetic and stoichiometric. Kinetic models can identify the control structure within a specific pathway, but they require highly detailed information, making them applicable to small sets of reactions. In contrast, stoichiometric models require only mass balance information, making them suitable for genome-scale modeling to study the effect of adding or removing reactions for the optimization of the synthesis of desired products. To study glycerol fermentation, a kinetic model was implemented, allowing prediction of the limiting enzymes of this process: glycerol dehydrogenase and di-hydroxyacetone kinase. This prediction was experimentally validated by increasing their enzymatic activities, resulting in a two-fold increase in the rate of ethanol production. Additionally, a stoichiometric genome-scale model (GEM) was modified to represent the fermentative metabolism of glycerol, identifying key metabolic pathways for glycerol fermentation (including a new glycerol dissimilation pathway). The GEM was used to identify genetic modifications that would increase the synthesis of desired products, such as succinate and butanol. Finally, glucose metabolism using the reversal β-oxidation cycle was modeled using a GEM to simulate the synthesis of a variety of medium and long chain products (including advanced biofuels). The model was used to design strategies that can lead to increase the productivity of target products.
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    Metabolic Engineering and Transhydrogenase Effects on NADPH Availability in Escherichia coli
    (2012-09-05) Jan, Joanna; San, Ka-Yiu; Bennett, George N.; Gonzalez, Ramon
    The ultimate goal in the field of metabolic engineering is improving cellular processes in a rational manner using engineering design principles and molecular biology techniques. The syntheses of several industrially useful compounds are cofactor-dependent. The reducing equivalent NADPH is required in several enzymatic reactions leading up to the synthesis of high-value compounds like polymers, chiral alcohols, and antibiotics. However, it’s a highly costly compound with limited intracellular availability. This study focuses on the genetic manipulation of a whole-cell system using the two transhydrogenase isoforms pntAB and udhA. Two model systems are used: 1) the production of (S)-2-chloropropionate and 2) the production of poly(3-hydroxybutyrate). Results suggest that the presence of udhA increases product yield and NADPH availability while the presence of pntAB has the opposite effect. A maximum product yield of 1.4 mole-product/mole-glucose was achieved aerobically in a pntAB-deletion strain with udhA overexpression, a 150% improvement over the wild-type control strain.
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    Metabolic flux analysis of fermentative carbon metabolism in Escherichia coli
    (2008) Murarka, Abhishek; Gonzalez, Ramon
    Recently, the production of various chemicals and fuels via microbial fermentation has gained momentum. Development of efficient bio-processes requires a system-scale understanding of the metabolic network of biocatalysts. In this context, we evaluated the roles of metabolic pathways and enzymes during fermentation of various substrates in E. coli using metabolic flux analysis (MFA), which is a powerful and efficient tool for comprehensive investigation of a biological system. The combination of substrates studied covered the full range of oxidation states of common carbon sources. During fermentation, pyruvate is a key precursor metabolite and a prominent intermediate for the synthesis of most fermentation products in E. coli. Under fermentative conditions, pyruvate is primarily dissimilated via pyruvate formate lyase (PFL), and pyruvate dehydrogenase (PDH) exhibits negligible activity with unknown physiological role. However, we found that the activity of PDH was required for efficient fermentative growth of E. coli. PDH was even able to support fermentative growth on glucuronate in a strain devoid of PFL. MFA indicated that a deletion of PDH leads to more than 10 fold increase in flux through oxidative pentose pathway. These results were supported by the 13C labeling based flux analysis. Subsequently, the hypothesized the role of PDH: to efficiently generate CO 2, assisting cell growth during glucose fermentation and to efficiently generate reducing equivalents aiding cell growth during glucuronate fermentation. In silico flux analysis was used to design further genetic modifications and supplementation experiments, which were instrumental in verifying our hypothesis. On the other hand, the fermentation of glycerol by E. coli, had been unknown until recently. In this study, we identified the factors facilitating this process in E. coli. Nuclear magnetic resonance (NMR) analysis of fermentation samples identified ethanol and 1,2-propanediol (1,2-PDO) as products of glycerol fermentation. Employing 13C tracer experiments, we demonstrated that majority of the fermentation products and about 20% of the biomass building blocks originate from glycerol. In silico flux analysis was instrumental in elucidating the role of active pathways during glycerol fermentation: redox-balanced pathway to ethanol generates energy for cell growth, and the redox consuming pathway synthesizing 1,2-PDO facilitates cell growth by enabling redox balance.