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Peter Czermak - One of the best experts on this subject based on the ideXlab platform.

  • Media development and process parameter optimization using statistical experimental designs for the production of nonribosomal peptides in Escherichia coli
    'Elsevier BV', 2021
    Co-Authors: Arne Michael Oestreich, Merlinda Ilire Suli, Doreen Gerlach, Rong Fan, Peter Czermak
    Abstract:

    Background: Nonribosomal peptide synthases (NRPS) can synthesize functionally diverse bioactive peptides by incorporating nonproteinogenic amino acids, offering a rich source of new drug leads. The bacterium Escherichia coli is a well-characterized production host and a promising candidate for the synthesis of nonribosomal peptides, but only limited Bioprocess Engineering has been reported for such molecules. We therefore developed a medium and optimized process parameters using the design of experiments (DoE) approach. Results: We found that glycerol is not suitable as a carbon source for rhabdopeptide production, at least for the NRPS used for this study. Alternative carbon sources from the tricarboxylic acid cycle achieved much higher yields. DoE was used to optimize the pH and temperature in a stirred-tank reactor, revealing that optimal growth and optimal production required substantially different conditions. Conclusions: We developed a chemically defined adapted M9 medium matching the performance of complex medium (lysogeny broth) in terms of product concentration. The maximum yield in the reactor under optimized conditions was 126 mg L-1, representing a 31-fold increase compared to the first shaking-flask experiments with M9 medium and glycerol as the carbon source. Conditions that promoted cell growth tended to inhibit NRPS productivity. The challenge was therefore to find a compromise between these factors as the basis for further process development.How to cite: Oestreich AM, Suli LI, Gerlach D. et al. Media development and process parameter optimization using statistical experimental designs for the production of nonribosomal peptides in Escherichia coli. Electron J Biotechnol 2021;52. https://doi.org/10.1016/j.ejbt.2021.05.00

  • three dimensional bioreactor technologies for the cocultivation of human mesenchymal stem stromal cells and beta cells
    Stem Cells International, 2018
    Co-Authors: Florian Petry, Tobias Weidner, Peter Czermak, Denise Salzig
    Abstract:

    Diabetes is a prominent health problem caused by the failure of pancreatic beta cells. One therapeutic approach is the transplantation of functional beta cells, but it is difficult to generate sufficient beta cells in vitro and to ensure these cells remain viable at the transplantation site. Beta cells suffer from hypoxia, undergo apoptosis, or are attacked by the host immune system. Human mesenchymal stem/stromal cells (hMSCs) can improve the functionality and survival of beta cells in vivo and in vitro due to direct cell contact or the secretion of trophic factors. Current cocultivation concepts with beta cells are simple and cannot exploit the favorable properties of hMSCs. Beta cells need a three-dimensional (3D) environment to function correctly, and the cocultivation setup is therefore more complex. This review discusses 3D cultivation forms (aggregates, capsules, and carriers) for hMSCs and beta cells and strategies for large-scale cultivation. We have determined process parameters that must be balanced and considered for the cocultivation of hMSCs and beta cells, and we present several bioreactor setups that are suitable for such an innovative cocultivation approach. Bioprocess Engineering of the cocultivation processes is necessary to achieve successful beta cell therapy.

  • mannheimia haemolytica growth and leukotoxin production for vaccine manufacturing a Bioprocess review
    Electronic Journal of Biotechnology, 2017
    Co-Authors: Peter Czermak, Tobias Oppermann, Nadine Busse
    Abstract:

    Mannheimia haemolytica leukotoxin (LKT) is a known cause of bovine respiratory disease (BRD) which results in severe economic losses in the cattle industry (up to USD 1 billion per year in the USA). Vaccines based on LKT offer the most promising measure to contain BRD outbreaks and are already commercially available. However, insufficient LKT yields, predominantly reflecting a lack of knowledge about the LKT expression process, remain a significant Engineering problem and further Bioprocess optimization is required to increase process efficiency. Most previous investigations have focused on LKT activity and cell growth, but neither of these parameters defines reliable criteria for the improvement of LKT yields. In this article, we review the most important process conditions and operational parameters (temperature, pH, substrate concentration, dissolved oxygen level, medium composition and the presence of metabolites) from a Bioprocess Engineering perspective, in order to maximize LKT yields. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0cm; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

  • Mannheimia haemolytica growth and leukotoxin production for vaccine manufacturing — A Bioprocess review
    Elsevier, 2017
    Co-Authors: Tobias Oppermann, Nadine Busse, Peter Czermak
    Abstract:

    Mannheimia haemolytica leukotoxin (LKT) is a known cause of bovine respiratory disease (BRD) which results in severe economic losses in the cattle industry (up to USD 1 billion per year in the USA). Vaccines based on LKT offer the most promising measure to contain BRD outbreaks and are already commercially available. However, insufficient LKT yields, predominantly reflecting a lack of knowledge about the LKT expression process, remain a significant Engineering problem and further Bioprocess optimization is required to increase process efficiency. Most previous investigations have focused on LKT activity and cell growth, but neither of these parameters defines reliable criteria for the improvement of LKT yields. In this article, we review the most important process conditions and operational parameters (temperature, pH, substrate concentration, dissolved oxygen level, medium composition and the presence of metabolites) from a Bioprocess Engineering perspective, in order to maximize LKT yields

Jin-byung Park - One of the best experts on this subject based on the ideXlab platform.

  • improving catalytic activity of the baeyer villiger monooxygenase based escherichia coli biocatalysts for the overproduction of z 11 heptanoyloxy undec 9 enoic acid from ricinoleic acid
    Scientific Reports, 2018
    Co-Authors: Jimin Woo, Eunyeong Jeon, Eunji Seo, Joohyun Seo, Dongyup Lee, Young Joo Yeon, Jin-byung Park
    Abstract:

    Baeyer-Villiger monooxygenases (BVMOs) can be used for the biosynthesis of lactones and esters from ketones. However, the BVMO-based biocatalysts are not so stable under process conditions. Thereby, this study focused on enhancing stability of the BVMO-based biocatalysts. The biotransformation of ricinoleic acid into (Z)-11-(heptanoyloxy)undec-9-enoic acid by the recombinant Escherichia coli expressing the BVMO from Pseudomonas putida and an alcohol dehydrogenase from Micrococcus luteus was used as a model system. After thorough investigation of the key factors to influence stability of the BVMO, Cys302 was identified as an Engineering target. The substitution of Cys302 to Leu enabled the engineered enzyme (i.e., E6BVMOC302L) to become more stable toward oxidative and thermal stresses. The catalytic activity of E6BVMOC302L-based E. coli biocatalysts was also greater than the E6BVMO-based biocatalysts. Another factor to influence biocatalytic performance of the BVMO-based whole-cell biocatalysts was availability of carbon and energy source during biotransformations. Glucose feeding into the reaction medium led to a marked increase of final product concentrations. Overall, the Bioprocess Engineering to improve metabolic stability of host cells in addition to the BVMO Engineering allowed us to produce (Z)-11-(heptanoyloxy)undec-9-enoic acid to a concentration of 132 mM (41 g/L) from 150 mM ricinoleic acid within 8 h.

  • Bioprocess Engineering to produce 9-(nonanoyloxy) nonanoic acid by a recombinant Corynebacterium glutamicum-based biocatalyst
    Journal of Industrial Microbiology & Biotechnology, 2017
    Co-Authors: Soohyun Park, Jeongmo Yang, Kijun Jeong, Jin-byung Park
    Abstract:

    Here, Corynebacterium glutamicum ATCC13032 expressing Baeyer–Villiger monooxygenase from Pseudomonas putida KT2440 was designed to produce 9-(nonanoyloxy) nonanoic acid from 10-ketostearic acid. Diverse parameters including cultivation and reaction temperatures, type of detergent, and pH were found to improve biotransformation efficiency. The optimal temperature of cultivation for the production of 9-(nonanoyloxy) nonanoic acid from 10-ketostearic acid using whole cells of recombinant C. glutamicum was 15 °C, but the reaction temperature was optimal at 30 °C. Enhanced conversion efficiency was obtained by supplying 0.05 g/L of Tween 80 at pH 7.5. Under these optimal conditions, recombinant C. glutamicum produced 0.28 mM of 9-(nonanoyloxy) nonanoic acid with a 75.6% (mol/mol) conversion yield in 2 h. This is the first report on the biotransformation of 10-ketostearic acid to 9-(nonanoyloxy) nonanoic acid with a recombinant whole-cell C. glutamicum -based biocatalyst and the results demonstrate the feasibility of using C. glutamicum as a whole-cell biocatalyst.

  • production of non proteinogenic amino acids from α keto acid precursors with recombinant corynebacterium glutamicum
    Biotechnology and Bioengineering, 2013
    Co-Authors: Ju-yeon Kim, Christoph Wittmann, Young-a Lee, Jin-byung Park
    Abstract:

    In the present work, Corynebacterium glutamicum was metabolically engineered for the enantioselective synthesis of non-proteinogenic amino acids as valuable building blocks for pharmaceuticals and agrochemicals. The novel bio-catalytic activity of C. glutamicum was obtained by heterologous expression of the branched chain aminotransferase IlvE from Escherichia coli. Upon this modification, the recombinant cells converted the α-keto acid precursor 2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid (HOAE) into the corresponding amino acid 2-(3-hydroxy-1-adamantyl)-(2S)-amino ethanoic acid (HAAE). Similarly, also L-tert-leucine could be obtained from trimethyl pyruvate indicating a broader applicability of the novel strategy. In both cases, the amino group donor glutamate was supplied from the endogenous metabolism of the recombinant producer. Hereby, the uptake of the precursor and secretion of the product was supported by an enhanced cell permeability through treatment of ethambutol, which inhibits arabinosyl transferases involved in cell wall biosynthesis. The excretion of HAAE into the reaction medium was linked to the secretion of glutamate, indicating a similar mechanism for the export of both compounds. On the other hand, the efflux of L-tert-leucine appeared to be driven by active transport. Subsequent Bioprocess Engineering enabled HAAE and L-tert-leucine to be produced at a rate of 0.21 and 0.42 mmol (g dry cells)⁻¹  h⁻¹, respectively up to a final product titer of 40 mM. Beyond the given examples, integrated metabolic and cell envelop Engineering might extend the production of a variety of other non-proteinogenic amino acids as well as chiral amines by C. glutamicum.

Marco Oldiges - One of the best experts on this subject based on the ideXlab platform.

  • FeedER: a feedback-regulated enzyme-based slow-release system for fed-batch cultivation in microtiter plates
    Bioprocess and Biosystems Engineering, 2019
    Co-Authors: Roman Jansen, Matthias Moch, Stephan Noack, Niklas Tenhaef, Wolfgang Wiechert, Marco Oldiges
    Abstract:

    With the advent of modern genetic Engineering methods, microcultivation systems have become increasingly important tools for accelerated strain phenotyping and Bioprocess Engineering. While these systems offer sophisticated capabilities to screen batch processes, they lack the ability to realize fed-batch processes, which are used more frequently in industrial Bioprocessing. In this study, a novel approach to realize a feedback-regulated enzyme-based slow-release system (FeedER), allowing exponential fed-batch for microscale cultivations, was realized by extending our existing Mini Pilot Plant technology with a customized process control system. By continuously comparing the experimental growth rates with predefined set points, the automated dosage of Amyloglucosidase enzyme for the cleavage of dextrin polymers into d -glucose monomers is triggered. As a prerequisite for stable fed-batch operation, a constant pH is maintained by automated addition of ammonium hydroxide. We show the successful application of FeedER to study fed-batch growth of different industrial model organisms including Corynebacterium   glutamicum , Pichia   pastoris , and Escherichia   coli . Moreover, the comparative analysis of a C.   glutamicum GFP producer strain, cultivated under microscale batch and fed-batch conditions, revealed two times higher product yields under slow growing fed-batch operation. In summary, FeedER enables to run 48 parallel fed-batch experiments in an automated and miniaturized manner, and thereby accelerates industrial Bioprocess development at the screening stage.

  • Application of metabolic Engineering for the biotechnological production of l-valine
    Applied Microbiology and Biotechnology, 2014
    Co-Authors: Marco Oldiges, Bernhard J. Eikmanns, Bastian Blombach
    Abstract:

    The branched chain amino acid l -valine is an essential nutrient for higher organisms, such as animals and humans. Besides the pharmaceutical application in parenteral nutrition and as synthon for the chemical synthesis of e.g. herbicides or anti-viral drugs, l -valine is now emerging into the feed market, and significant increase of sales and world production is expected. In accordance, well-known microbial production bacteria, such as Escherichia coli and Corynebacterium glutamicum strains, have recently been metabolically engineered for efficient l -valine production under aerobic or anaerobic conditions, and the respective cultivation and production conditions have been optimized. This review summarizes the state of the art in l -valine biosynthesis and its regulation in E. coli and C. glutamicum with respect to optimal metabolic network for microbial l -valine production, genetic strain Engineering and Bioprocess development for l -valine production, and finally, it will shed light on emerging technologies that have the potential to accelerate strain and Bioprocess Engineering in the near future.

Joaquim M. S. Cabral - One of the best experts on this subject based on the ideXlab platform.

  • scalable microcarrier based manufacturing of mesenchymal stem stromal cells
    Journal of Biotechnology, 2016
    Co-Authors: Antonio M De Soure, Ana Fernandesplatzgummer, Claudia Lobato Da Silva, Joaquim M. S. Cabral
    Abstract:

    Due to their unique features, mesenchymal stem/stromal cells (MSC) have been exploited in clinical settings as therapeutic candidates for the treatment of a variety of diseases. However, the success in obtaining clinically-relevant MSC numbers for cell-based therapies is dependent on efficient isolation and ex vivo expansion protocols, able to comply with good manufacturing practices (GMP). In this context, the 2-dimensional static culture systems typically used for the expansion of these cells present several limitations that may lead to reduced cell numbers and compromise cell functions. Furthermore, many studies in the literature report the expansion of MSC using fetal bovine serum (FBS)-supplemented medium, which has been critically rated by regulatory agencies. Alternative platforms for the scalable manufacturing of MSC have been developed, namely using microcarriers in bioreactors, with also a considerable number of studies now reporting the production of MSC using xenogeneic/serum-free medium formulations. In this review we provide a comprehensive overview on the scalable manufacturing of human mesenchymal stem/stromal cells, depicting the various steps involved in the process from cell isolation to ex vivo expansion, using different cell tissue sources and culture medium formulations and exploiting Bioprocess Engineering tools namely microcarrier technology and bioreactors.

  • a xenogeneic free bioreactor system for the clinical scale expansion of human mesenchymal stem stromal cells
    Biotechnology and Bioengineering, 2014
    Co-Authors: Francisco Dos Santos, Ana Fernandesplatzgummer, Claudia Lobato Da Silva, Andrew Campbell, Pedro Z Andrade, Jeffrey M Gimble, Yuan Wen, Shayne Boucher, Mohan C Vemuri, Joaquim M. S. Cabral
    Abstract:

    The large cell doses (>1 × 10(6)  cells/kg) used in clinical trials with mesenchymal stem/stromal cells (MSC) will require an efficient production process. Moreover, monitoring and control of MSC ex-vivo expansion is critical to provide a safe and reliable cell product. Bioprocess Engineering approaches, such as bioreactor technology, offer the adequate tools to develop and optimize a cost-effective culture system for the rapid expansion of human MSC for cellular therapy. Herein, a xenogeneic (xeno)-free microcarrier-based culture system was successfully established for bone marrow (BM) MSC and adipose tissue-derived stem/stromal cell (ASC) cultivation using a 1L-scale controlled stirred-tank bioreactor, allowing the production of (1.1 ± 0.1) × 10(8) and (4.5 ± 0.2) × 10(7) cells for BM MSC and ASC, respectively, after 7 days. Additionally, the effect of different percent air saturation values (%Airsat ) and feeding regime on the proliferation and metabolism of BM MSC was evaluated. No significant differences in cell growth and metabolic patterns were observed under 20% and 9%Airsat . Also, the three different feeding regimes studied-(i) 25% daily medium renewal, (ii) 25% medium renewal every 2 days, and (iii) fed-batch addition of concentrated nutrients and growth factors every 2 days-yielded similar cell numbers, and only slight metabolic differences were observed. Moreover, the immunophenotype (positive for CD73, CD90 and CD105 and negative for CD31, CD80 and HLA-DR) and multilineage differentiative potential of expanded cells were not affected upon bioreactor culture. These results demonstrated the feasibility of expanding human MSC from different sources in a clinically relevant expansion configuration in a controlled microcarrier-based stirred culture system under xeno-free conditions. The further optimization of this bioreactor culture system will represent a crucial step towards an efficient GMP-compliant clinical-scale MSC production system.

  • scalable ex vivo expansion of human mesenchymal stem stromal cells in microcarrier based stirred culture systems
    Methods of Molecular Biology, 2014
    Co-Authors: Joana G. Carmelo, Ana Fernandesplatzgummer, Joaquim M. S. Cabral, Claudia Lobato Da Silva
    Abstract:

    : The clinical demand for human mesenchymal stem/stromal cells (MSC) drives the need for reproducible, cost-effective, and good manufacturing practices (GMP)-compliant ex vivo expansion protocols. Bioprocess Engineering strategies, namely controlled stirred bioreactor systems combined with the use of xenogeneic(xeno)-free materials, provide proper tools to develop and optimize cell manufacturing for the rapid expansion of human MSC for cellular therapies. Herein we describe a microcarrier-based stirred culture system operating under xeno-free conditions using a controlled stirred-tank bioreactor for an efficient and controlled ex vivo expansion of human MSC. This culture platform can be applied to MSC from different human sources, as well as different microcarriers and xeno-free medium formulations.

Yadollah Omidi - One of the best experts on this subject based on the ideXlab platform.

  • Bioprocess Engineering of echium italicum l induction of shikonin and alkannin derivatives by two liquid phase suspension cultures
    Plant Cell Tissue and Organ Culture, 2010
    Co-Authors: Khadijeh Zare, Hossein Nazemiyeh, Ali Movafeghi, Mahmood Khosrowshahli, Alireza Motallebiazar, Mohammadreza Dadpour, Yadollah Omidi
    Abstract:

    An in vitro cell suspension culture of Echium italicum was established and assayed for the production of shikonin and alkannin derivatives. Callus tissues were induced from cotyledon explants of the plant incubated onto the solidified B5 medium. A two-liquid-phase system suspension culture was then established to elicit pigments of shikonin and alkannin derivatives using liquid paraffin. The presence of liquid paraffin efficiently induced production of pigments in cultured cells. The production and/or accumulation of these compounds in the E. italicum cells was examined using fluorescence microscopy as the naphthoquinone molecules display autofluorescent properties. Phytochemical analysis of the n-hexane extract of the medium was also carried out using preparative HPLC. The chemical structure of shikonin and alkannin derivatives were characterized by UV, 1H-NMR, and 13C-NMR techniques. Based on our findings, this Bioprocess Engineering approach resulted in induction of shikonin and alkannin derivatives, whereupon it may be recruited for production of these important secondary metabolites.

  • rapid and simple methodology for isolation of high quality genomic dna from coniferous tissues taxus baccata
    Molecular Biology Reports, 2010
    Co-Authors: Abolfazl Barzegari, Sepideh Zununi Vahed, Sina Atashpaz, Sajjad Khani, Yadollah Omidi
    Abstract:

    Various investigations have been so far performed for extraction of genomic DNA from plant tissues, in which the extracted intact DNA can be exploited for a diverse range of biological studies. Extraction of high quality DNA from leathery plant tissues (e.g., coniferous organs) appears to be a critical stage. Moreover, for some species such as Taxus trees, Bioprocess Engineering and biosynthesis of secondary metabolites (e.g., paclitaxel) is a crucial step due to the restrictions associated with extinction of these species. However, extraction of intact genomic DNA from these plants still demands a rapid, easy and efficient protocol. To pursue such aim, in the current work, we report on the development of a simple and highly efficient method for the extraction of DNA from Taxus baccata. Based upon our protocol, interfering phenolic compounds were removed from extraction using polyvinylpyrrolidone and RNA contamination was resolved using LiCl. By employing this method, high quality genomic DNA was successfully extracted from leaves of T. baccata. The quality of extracted DNA was validated by various techniques such as RAPD marker, restriction digestions and pre-AFLP. Upon our findings, we propose this simple method to be considered for extraction of DNA from leathery plant tissues.

  • improved taxol production by combination of inducing factors in suspension cell culture of taxus baccata
    Cell Biology International, 2006
    Co-Authors: Yari A Khosroushahi, Mahmood Khosrowshahli, Mohammadreza Dadpour, Mostafa Valizadeh, A R Ghasempour, H A Naghdibadi, Yadollah Omidi
    Abstract:

    Abstract To date enormous attempts have been devoted to improve Taxol production exploiting various methodologies from Bioprocess Engineering to biotechnological and synthetic approaches. We have developed a 2-stage suspension cell culture of Taxus baccata L. using modified B5 medium in order to improve cell growth as well as productivity. After callus induction and cell line selection, B5 medium was supplemented with vanadyl sulfate (0.1 mg/l), silver nitrate (0.3 mg/l) and cobalt chloride (0.25 mg/l) at the first day of stage I culture to maximize cell growth. This medium was further supplemented with sucrose (1%) and ammonium citrate (50 mg/l) on day 10 and sucrose (1%) and phenylalanine (0.1 mM) on day 20 (i.e., biomass growth medium). At stage II (day 25), two different concentrations of several elicitors such as methyl jasmonate (10 or 20 mg/l), salicylic acid (50 or 100 mg/l) and fungal elicitor (25 or 50 mg/l) were added to the biomass growth medium with the aim of improving cellular productivity. For morphological analysis, microscopic inspection was carried out during cultivation. Cell-associated and extracellular amount of Taxol were detected and measured using HPLC methodology. At stage I, overall Taxol amount of biomass growth medium was 13.75 mg/l (i.e., 5.6-fold higher than that of untreated B5 control). At stage II, treated cells with methyl jasmonate (10 mg/l), salicylic acid (100 mg/l) and fungal elicitor (25 mg/l) produced the highest amount of Taxol (39.5 mg/l), which is 16-fold higher than that of untreated B5 control (2.45 mg/l). Microscopic analyses of Taxus cells in suspension cultures showed various positional auto-fluorescence showing direct correlation with Taxol production. Our studies revealed that intervallic supplementation of B5 medium with combination of biomass growth factors at stage I and mixture of elicitors at stage II could significantly increase Taxol production. Thus, we suggest that the exploitation of this methodology may improve the production of Taxol since demands for Taxol pharmaceuticals are increasingly growing and resource paucities have limited its direct harvesting from Taxus trees.