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  • 1.
    Faisal, Abrar
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Holmlund, Mattias
    Swedish University of Agriculture Sciences.
    Ginesy, Mireille
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Holmgren, Allan
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Enman, Josefine
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Hedlund, Jonas
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Grahn, Mattias
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Recovery of l-Arginine from Model Solutions and Fermentation Broth Using Zeolite-Y Adsorbent2019In: ACS Sustainable Chemistry and Engineering, E-ISSN 2168-0485, Vol. 7, no 9, p. 8900-8907Article in journal (Refereed)
    Abstract [en]

    Arginine was produced via fermentation of sugars using the engineered microorganism Escherichia coli. Zeolite-Y adsorbents in the form of powder and extrudates were used to recover arginine from both a real fermentation broth and aqueous model solutions. An adsorption isotherm was determined using model solutions and zeolite-Y powder. The saturation loading was determined to be 0.2 g/g using the Sips model. Arginine adsorbed from a real fermentation broth using either zeolite-Y powder or extrudates both showed a maximum loading of 0.15 g/g at pH 11. This adsorbed loading is very close to the corresponding value obtained from the model solution showing that under the experimental conditions the presence of additional components in the broth did not have a significant effect on the adsorption of arginine. Furthermore, a breakthrough curve was determined for extrudates using a 1 wt % arginine model solution. The selectivity for arginine over ammonia and alanine from the real fermentation broth at pH 11 was 1.9 and 8.3, respectively, for powder, and 1.0, and 4.1, respectively, for extrudates. To the best of our knowledge, this is the first time recovery of arginine from real fermentation broths using any type of adsorbent has been reported.

  • 2.
    Ginesy, Mireille
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Production of L-arginine by Escherichia coli: Impact of genetic modifications, carbon and nitrogen sources2021Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In the recent years, the demand for environmental friendly produced L-arginine has risen with the increasing number of applications for this amino acid in pharmaceuticals, nutraceuticals, cosmetics, animal feed and fertilizers.Microbial production of L-arginine usually relies on Corynebacterium glutam-icum and Corynebacterium crenatum strains. However, Escherichia coli presents the advantage of being able to utilize a wider range of substrates, including pentose sugars found in lignocellulosic feedstocks.The present thesis illustrates the first steps in the development of a sustain-able process to produce L-arginine using E. coli. It starts with the construction of an L-arginine overproducing strain, followed by an investigation into adequate nitrogen and carbon sources for cell growth and L-arginine production.The first part of this thesis aimed at engineering an E. coli strain able to produce high level of L-arginine. Mutations on key genes of the L-arginine biosynthesis pathway were stepwisely done. The mutants obtained at each step were tested in bioreactor fermentations to assess the effect of each genetic modification. The final strain was able to produce almost 12 g/L during fer-mentation, at a productivity of 0.24 g/L/h. In comparison, the starting strain, E. coli K12 C600, was not able to excrete any L-arginine. Besides, one mutant from each step was further examined in a metabolomic study in order to gain deeper insight into the effects of the various genetic modifications performed.To minimize nitrogen waste and optimize the L-arginine production the impact of different nitrogen sources and concentrations were then investigated. Ammonium phosphate dibasic, ammonium sulfate and ammonia solution were the best nitrogen sources for L-arginine production. In minimal medium, the optimum carbon to nitrogen ratio was 6, yielding about 4 g/L L-arginine from 30 g/L glucose. At this ratio, both glucose and the nitrogen source were com-pletely utilized during fermentation.Finaly various carbon courses commonly found in lignocellulosic feedstocks were tested. D-glucose and D-xylose were the most suitable carbon sources followed by L-arabinose. D-galactose and D-mannose resulted in significantly less arginie formation. However, a mixture of all five sugars yielded a higher production than any individual sugar.

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  • 3.
    Ginesy, Mireille
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Production of L-arginine by genetically modified Escherichia coli2015Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    In the recent years, the demand for environmental friendly produced L-arginine has risen with the increasing number of applications for this amino acid in pharmaceuticals, nutraceuticals, cosmetics, animal feed and fertilizers. Members of the Corynebacteriaceae family are usually used for microbial L-arginine production. However, Escherichia coli present the advantage of being able to utilize a wider range of substrates, including pentose sugars found in lignocellulosic feedstocks. The present thesis illustrates the first steps in the development of a sustainable process to produce L-arginine using E. coli. It starts with the constructions of a L-arginine overproducing strain, followed by optimization of the nitrogen supply for the fermentations.The first part of this thesis aimed at engineering an E. coli train able to produce high level of L-arginine. Mutations on key genes of the L-arginine biosynthesis pathway were step-wisely done. The mutants obtained at each step were tested in bioreactor fermentations to assess the effect of each genetic modification. The final strain was able to produce almost 12 g/l during fermentation, at a productivity of 0.24 g/l/h. In comparison the starting strain, E. coli K12 C600, was not able to excrete any L-arginine. To minimize nitrogen wastes and optimize the L-arginine production the impact of different nitrogen sources and concentration were investigated. It was shown that while ammonium phosphate dibasic was the most potent nitrogen source during cultivation on complex medium, all the sources were equivalent with minimal media; this probably reflected the phosphate deficiency of the complex medium used. In fermentation on minimal medium, a carbon to nitrogen ratio of 5 was demonstrated to be the most suitable, yielding up to 4.5 g/l L-arginine. At this ratio, both glucose and the nitrogen source were completely utilized during fermentation.

  • 4.
    Ginesy, Mireille
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Belotserkovsky, Jaroslav
    Department of Molecular Biosciences Wenner-Gren institute, Stockholm University.
    Enman, Josefine
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Isaksson, Leif
    Department of Molecular Biosciences Wenner-Gren institute, Stockholm University.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Metabolic engineering of Escherichia coli for enhanced arginine biosynthesis2015In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, no 1, article id 29Article in journal (Refereed)
    Abstract [en]

    BackgroundArginine is a high-value product, especially for the pharmaceutical industry. Growing demand for environmental-friendly and traceable products have stressed the need for microbial production of this amino acid. Therefore, the aim of this study was to improve arginine production in Escherichia coli by metabolic engineering and to establish a fermentation process in 1-L bioreactor scale to evaluate the different mutants. ResultsFirstly, argR (encoding an arginine responsive repressor protein), speC, speF (encoding ornithine decarboxylases) and adiA (encoding an arginine decarboxylase) were knocked out and the feedback-resistant argA214 or argA215 were introduced into the strain. Three glutamate independent mutants were assessed in bioreactors. Unlike the parent strain, which did not excrete any arginine during glucose fermentation, the constructs produced between 1.94 and 3.03 g/L arginine. Next, wild type argA was deleted and the gene copy number of argA214 was raised, resulting in a slight increase in arginine production (4.11 g/L) but causing most of the carbon flow to be redirected toward acetate. The V216A mutation in argP (transcriptional regulator of argO, which encodes for an arginine exporter) was identified as a potential candidate for improved arginine production. The combination of multicopy of argP216 or argO and argA214 led to nearly 2-fold and 3-fold increase in arginine production, respectively, and a reduction of acetate formation. ConclusionsIn this study, E. coli was successfully engineered for enhanced arginine production. The ∆adiA, ∆speC, ∆speF, ∆argR, ∆argA mutant with high gene copy number of argA214 and argO produced 11.64 g/L of arginine in batch fermentation, thereby demonstrating the potential of E. coli as an industrial producer of arginine.

  • 5.
    Ginesy, Mireille
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Enman, Josefine
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rusanova-Naydenova, Daniela
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Simultaneous Quantification of L-Arginine and Monosaccharides during Fermentation: An Advanced Chromatography Approach2019In: Molecules, ISSN 1431-5157, E-ISSN 1420-3049, Vol. 24, no 4, article id 802Article in journal (Refereed)
    Abstract [en]

    Increasing demand for L-arginine by the food and pharmaceutical industries has sparked the search for sustainable ways of producing it. Microbial fermentation offers a suitable alternative; however, monitoring of arginine production and carbon source uptake during fermentation, requires simple and reliable quantitative methods compatible with the fermentation medium. Two methods for the simultaneous quantification of arginine and glucose or xylose are described here: high-performance anion-exchange chromatography coupled to integrated pulsed amperometric detection (HPAEC-IPAD) and reversed-phase ultra-high-performance liquid chromatography combined with charged aerosol detection (RP-UHPLC-CAD). Both were thoroughly validated in a lysogeny broth, a minimal medium, and a complex medium containing corn steep liquor. HPAEC-IPAD displayed an excellent specificity, accuracy, and precision for arginine, glucose, and xylose in minimal medium and lysogeny broth, whereas specificity and accuracy for arginine were somewhat lower in medium containing corn steep liquor. RP-UHPLC-CAD exhibited high accuracy and precision, and enabled successful monitoring of arginine and glucose or xylose in all media. The present study describes the first successful application of the above chromatographic methods for the determination and monitoring of L-arginine amounts during its fermentative production by a genetically modified Escherichia coli strain cultivated in various growth media.

  • 6.
    Ginesy, Mireille
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rusanova-Naydenova, Daniela
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Tuning of the Carbon-to-Nitrogen Ratio for the Production of L-Arginine by Escherichia coli2017In: Fermentation, ISSN 2311-5637, Vol. 3, no 4, article id 60Article in journal (Refereed)
    Abstract [en]

    L-arginine, an amino acid with a growing range of applications within the pharmaceutical, cosmetic, food, and agricultural industries, can be produced by microbial fermentation. Although it is the most nitrogen-rich amino acid, reports on the nitrogen supply for its fermentation are scarce. In this study, the nitrogen supply for the production of l-arginine by a genetically modified Escherichia coli strain was optimised in bioreactors. Different nitrogen sources were screened and ammonia solution, ammonium sulphate, ammonium phosphate dibasic, and ammonium chloride were the most favourable nitrogen sources for l-arginine synthesis. The key role of the C/N ratio for l-arginine production was demonstrated for the first time. The optimal C/N molar ratio to maximise l-arginine production while minimising nitrogen waste was found to be 6, yielding approximately 2.25 g/L of l-arginine from 15 g/L glucose with a productivity of around 0.11 g/L/h. Glucose and ammonium ion were simultaneously utilized, showing that this ratio provided a well-balanced equilibrium between carbon and nitrogen metabolisms.

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1 - 6 of 6
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