Biotechnology and Bioengineering

Cover image for Vol. 110 Issue 9

September 2013

Volume 110, Issue 9

Pages C1–C1, fmi–fmvi, 2317–2561

  1. Cover

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlights
    5. Viewpoint
    6. Articles
    7. Communication to the Editor
  2. Contents

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlights
    5. Viewpoint
    6. Articles
    7. Communication to the Editor
  3. Spotlights

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlights
    5. Viewpoint
    6. Articles
    7. Communication to the Editor
  4. Viewpoint

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlights
    5. Viewpoint
    6. Articles
    7. Communication to the Editor
    1. Viewpoint

      You have free access to this content
      A matter of detail: Assessing the true potential of microalgal biofuels (pages 2317–2322)

      Daniel Klein-Marcuschamer, Yusuf Chisti, John R. Benemann and David Lewis

      Version of Record online: 21 JUN 2013 | DOI: 10.1002/bit.24967

  5. Articles

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlights
    5. Viewpoint
    6. Articles
    7. Communication to the Editor
    1. Biocatalysis, Protein Engineering, and Nanobiotechnology

      Oxyfunctionalization of aliphatic compounds by a recombinant peroxygenase from Coprinopsis cinerea (pages 2323–2332)

      Esteban D. Babot, José C. del Río, Lisbeth Kalum, Angel T. Martínez and Ana Gutiérrez

      Version of Record online: 22 APR 2013 | DOI: 10.1002/bit.24904

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      Coprinopsis cinerea peroxygenase, produced as a recombinant (rCcP), selectively hydroxylates long-chain alkanes, fatty alcohols, and free/esterified fatty acids at two subterminal positions under mild conditions using H2O2 as the sole co-substrate.

    2. Investigations on diffusion limitations of biocatalyzed reactions in amphiphilic polymer conetworks in organic solvents (pages 2333–2342)

      Ina Schoenfeld, Stephan Dech, Benjamin Ryabenky, Bastian Daniel, Britta Glowacki, Reinhild Ladisch and Joerg C. Tiller

      Version of Record online: 14 MAY 2013 | DOI: 10.1002/bit.24906

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      Enzymes entrapped in amphiphilic polymer conetworks (APCNs) show greatly increased activity in organic solvents. Unfortunately, these reactions suffer from diffusion limitations. In this article, different APCNs membranes loaded with Rizomucor meihei lipase and chymotrypsin were particulized, obtaining microparticles with 5–10 µm in size. Although the reactions are still diffusion limited within these particles, the entrapped enzymes are highly activated compared to the membranes and even compared to commercial systems.

    3. Effects of pre-existing anti-carrier immunity and antigenic element multiplicity on efficacy of a modular virus-like particle vaccine (pages 2343–2351)

      Yap P. Chuan, Tania Rivera-Hernandez, Nani Wibowo, Natalie K. Connors, Yang Wu, Fiona K. Hughes, Linda H.L. Lua and Anton P.J. Middelberg

      Version of Record online: 22 APR 2013 | DOI: 10.1002/bit.24907

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      Modularization of a peptide antigen for presentation on a microbially-synthesized murine polyomavirus (MuPyV) virus-like particle (VLP) offers new alternatives for rapid and low-cost vaccine delivery at a global scale. Immunization with a modular MuPyV VLP presenting an antigenic element from group A streptococcus (GAS) induced high levels of antibodies against the element despite a strong pre-existing anti-carrier immune response. This study also demonstrated the possibility of modularizing the VLP to present up to 720 copies of the antigenic element.

    4. Stabilization of enzymes in ionic liquids via modification of enzyme charge (pages 2352–2360)

      Erik M. Nordwald and Joel L. Kaar

      Version of Record online: 22 APR 2013 | DOI: 10.1002/bit.24910

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      A strategy to broadly improve enzyme utility in ionic liquids was developed based on elucidating the effect of charge modifications on enzyme function in ionic liquids. Results of stability studies found that modifications that reduce the ratio of enzyme-containing positive-to-negative sites greatly improve enzyme stability in ionic liquids. Understanding the impact of charge modification on enzyme stability may ultimately be exploited to rationally engineer enzymes for improved function in ionic liquid environments.

    5. Controlling enzyme inhibition using an expanded set of genetically encoded amino acids (pages 2361–2370)

      Shun Zheng and Inchan Kwon

      Version of Record online: 22 APR 2013 | DOI: 10.1002/bit.24911

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      Replacement of a key residue for an inhibitor binding with a non-natural amino acid containing a bulkier side chain restricts the inhibitor binding. Here the authors show that replacing a key phenylalanine (Phe) residue for inhibitor methotrexate (MTX) binding of murine dihydrofolate reductase (mDHFR) enzyme with two non-natural amino acids (p-bromophenylalanine (pBrF) and L-2-naphthylalanine (2Nal) enhances binding affinity toward the substrate over the inhibitor by 4.0 and 5.8 times, respectively. This is mainly due to a reduced inhibitor binding affinity by 2.1 and 4.3 times, respectively. The work described here clearly demonstrates the feasibility of controlling enzyme inhibition using an expanded set of genetically encoded amino acids.

    6. Biofuels and Environmental Biotechnology

      Rhythm of carbon and nitrogen fixation in unicellular cyanobacteria under turbulent and highly aerobic conditions (pages 2371–2379)

      S. Krishnakumar, Sandeep B. Gaudana, Ganesh A. Viswanathan, Himadri B. Pakrasi and Pramod P. Wangikar

      Version of Record online: 7 APR 2013 | DOI: 10.1002/bit.24882

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      The authors show that Cyanothece sp. ATCC 51142 grows well and fixes nitrogen under highly turbulent and aerobic conditions. Turbulent regime ensures rapid movement of cells between light and dark zones and creates a flashing light effect. A respiratory burst around dusk creates intracellular anoxic conditions thereby protecting the nitrogenase enzyme from molecular oxygen. Magnitudes of the respiratory bursts in terms of carbon dioxide or oxygen evolution rates correlate well with growth rate at different agitation speeds (rpm).

    7. Development and evaluation of methods to infer biosynthesis and substrate consumption in cultures of cellulolytic microorganisms (pages 2380–2388)

      Evert K. Holwerda, Lucas D. Ellis and Lee R. Lynd

      Version of Record online: 30 APR 2013 | DOI: 10.1002/bit.24915

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      Concentrations of biosynthate (microbial biomass plus extracellular proteins) and residual substrate were determined for batch cultures of Clostridium thermocellum growing on cellobiose and cellulose using elemental analysis. This was compared to different on-line measurements: base addition, CO2-production and Near Infra Red optical density (OD850). On cellulose both biosynthate and residual substrate concentration demonstrate typical sigmoidal trends. The instantaneous specific growth rate was determined; while soluble substrate grown cultures show a constant growth rate, cultures grown on solid substrate do not.

    8. Testing alternative kinetic models for utilization of crystalline cellulose (Avicel) by batch cultures of Clostridium thermocellum (pages 2389–2394)

      Evert K. Holwerda and Lee R. Lynd

      Version of Record online: 29 APR 2013 | DOI: 10.1002/bit.24914

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      Three models were tested to describe the kinetics of (Avicel) utilization by Clostridium thermocellum: (A) first order in cells, (B) first order in substrate, and (C) first order in cells and substrate; second order overall. Model (C) provided by far the best fit to batch culture data. A second order rate constant (0.735 L g C−1 h−1) was found for utilization of (Avicel) by C. thermocellum. Adding an endogenous metabolism term improved the descriptive quality of the model as substrate exhaustion was approached.

    9. Improvement of ethanol productivity and energy efficiency by degradation of inhibitors using recombinant Zymomonas mobilis (pHW20a-fdh) (pages 2395–2404)

      Hong-Wei Dong, Li-Qiang Fan, Zichen Luo, Jian-Jiang Zhong, Dewey D. Y. Ryu and Jie Bao

      Version of Record online: 5 JUL 2013 | DOI: 10.1002/bit.24897

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      Scheme of proposed simultaneous detoxification of formate and furan compounds: A recombinant Z. mobilis ZM4 (pHW20a-fdh) strain, which is capable of degrading toxic formate, is constructed according to the scheme shown in the figure. This is accomplished by cloning heterologous formate dehydrogenase gene (fdh) from Saccharomyces cerevisiae and by coupling this reaction of the NADH regeneration reaction system with furfural and HMF degradation in the recombinant Z. mobilis strain. ADH II, alcohol dehydrogenase; FDH, formate dehydrogenase.

    10. Development of a novel electrochemical system for oxygen control (ESOC) to examine dissolved oxygen inhibition on algal activity (pages 2405–2411)

      Philip C. Keymer, Steven Pratt and Paul A. Lant

      Version of Record online: 1 JUL 2013 | DOI: 10.1002/bit.24905

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      A novel Electrochemical System for Oxygen Control (ESOC), which relies on coulombic titration to balance the electrochemical reduction of oxygen with oxygen input to achieve a steady DO setpoint, has been developed for examining algal photosynthetic activity as a function of dissolved oxygen (DO). Application of the tool for the study of five Scenedesmus algal systems shows that algal activity is inhibited at elevated DO, and that inhibition can be described by a Hill inhibition model.

    11. Bioprocess Engineering and Supporting Technologies

      Bioreactor engineering using disposable technology for enhanced production of hCTLA4Ig in transgenic rice cell cultures (pages 2412–2424)

      Jun-Young Kwon, Yong-Suk Yang, Su-Hwan Cheon, Hyung-Jin Nam, Gi-Hong Jin and Dong-Il Kim

      Version of Record online: 22 APR 2013 | DOI: 10.1002/bit.24916

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      Two kinds of disposable bioreactors, air-lift disposable bioreactors (ADB) and Wave disposable bioreactors (WDB) were successfully applied to transgenic rice cell cultures for the production of recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). To manipulate the dimensions in ADB and WDB, disposable technology was used and it will be useful for development and scale-up studies of disposable bioreactor systems for transgenic plant cell cultures.

    12. Bioseparations and Downstream Processing

      You have full text access to this OnlineOpen article
      Fouling of an anion exchange chromatography operation in a monoclonal antibody process: Visualization and kinetic studies (pages 2425–2435)

      Edward J. Close, Jeffrey R. Salm, Timothy Iskra, Eva Sørensen and Daniel G. Bracewell

      Version of Record online: 31 MAR 2013 | DOI: 10.1002/bit.24898

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      Scanning electron microscopy (SEM), batch uptake experiments, confocal laser scanning microscopy (CLSM) and small-scale column experiments were applied to characterize a case study where fouling had been observed on an anion exchange chromatography in a monoclonal antibody process. The results suggest the foulant is located on the particle surface, resulting in a minimal decrease in saturation capacity, but having a significant impact on the kinetics of adsorption, severely decreasing protein uptake rate.

    13. multifraction separation in countercurrent chromatography (MCSGP) (pages 2436–2444)

      Martin Krättli, Thomas Müller-Späth and Massimo Morbidelli

      Version of Record online: 22 APR 2013 | DOI: 10.1002/bit.24901

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      The MCSGP process known for the purification of biomolecules is extended to separate multiple pure fractions at once. The concept is experimentally verified applying a protein model system and a monoclonal antibody.

    14. Protein a chromatography at high titers (pages 2445–2451)

      Venkatesh Natarajan and Andrew L. Zydney

      Version of Record online: 22 APR 2013 | DOI: 10.1002/bit.24902

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      Dynamic binding capacity decreases at high antibody titers and short residence times.

    15. Fast and scalable purification of a therapeutic full-length antibody based on process crystallization (pages 2452–2461)

      Benjamin Smejkal, Neeraj J. Agrawal, Bernhard Helk, Henk Schulz, Marion Giffard, Matthias Mechelke, Franziska Ortner, Philipp Heckmeier, Bernhardt L. Trout and Dariusch Hekmat

      Version of Record online: 22 APR 2013 | DOI: 10.1002/bit.24908

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      The potential of process crystallization for purification of a therapeutic full-length monoclonal antibody was studied. It was shown that crystallization has a strong potential to replace Protein A chromatography. A reproducible stirred crystallization process from pretreated harvest was established and scaled-up to the 1 L-scale. High yield, high purity, and high biological activity was achieved. Furthermore, molecular modeling suggested that a negative electrostatic region with interspersed exposed hydrophobic residues is responsible for the crystallization propensity of this antibody.

    16. Model-based risk analysis of coupled process steps (pages 2462–2470)

      Karin Westerberg, Ernst Broberg-Hansen, Lars Sejergaard and Bernt Nilsson

      Version of Record online: 22 APR 2013 | DOI: 10.1002/bit.24909

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      A risk analysis assessing the impact of process parameter variations on the product quality is essential in biopharmaceutical process development. A process model is used to make the parameter study and guide the experiment plan for a process with added complexity due to recirculation. The experimental effort was reduced while process understanding was increased.

    17. Split intein mediated ultra-rapid purification of tagless protein (SIRP) (pages 2471–2481)

      Dongli Guan, Miguel Ramirez and Zhilei Chen

      Version of Record online: 22 APR 2013 | DOI: 10.1002/bit.24913

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      Tag removal poses a significant challenge in recombinant protein purification. The authors describe an engineered split intein for ultra-rapid affinity tag removal. This system exhibits extraordinarily rapid thio-induced C-terminal cleavage with about 50% completions within 30 s at both 22°C and 6°C.Target protein is cleaved to >90% completion in 30 minutes at room temperature, with minimal sensitivity to the identity of the N-terminal amino acid. This technology should provide a useful tool for the purification of tagless proteins and peptides.

    18. Cellular and Metabolic Engineering

      Inverse metabolic engineering to improve Escherichia coli as an N-glycosylation host (pages 2482–2493)

      Jagroop Pandhal, Lauren B. A. Woodruff, Stephen Jaffe, Pratik Desai, Saw Y. Ow, Josselin Noirel, Ryan T. Gill and Phillip C. Wright

      Version of Record online: 17 MAY 2013 | DOI: 10.1002/bit.24920

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      Inverse metabolic engineering to improve the efficiency of E. coli as an N-glycosylation host is described. Genetic elements, native to E. coli, which improved N-glycosylation of AcrA, were identified using a glycan-specific screen and mapped against the E. coli W3110 genome (circular outline). Colored lines represent genes sourced from different sized libraries. The charts show absolute quantification of total AcrA, glycosylated AcrA and also glycosylation efficiency, for strains forward engineered to overexpress the genetic elements labeled on the genome.

    19. Candida bombicola as a platform organism for the production of tailor-made biomolecules (pages 2494–2503)

      Sophie L.K.W. Roelants, Karen M.J. Saerens, Thibaut Derycke, Bing Li, Yao-Cheng Lin, Yves Van de Peer, Sofie L. De Maeseneire, Inge N.A. Van Bogaert and Wim Soetaert

      Version of Record online: 1 MAY 2013 | DOI: 10.1002/bit.24895

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      The industrially important yeast Candida bombicola produces high amounts (more than 400 g/L) of the biosurfactant molecules sophorolipids, which have already found applications in cosmetics and ecological cleaning solutions. In this work, proof of concept was delivered that this highly productive yeast can be transformed into a platform organism for the production of interesting (new-to-nature) biomolecules. Two examples were provided; the bioplastic PHA and a new to nature cellobioselipid were successfully produced by two genetically engineered strains.

    20. Analysis and comparison of oxygen consumption of HepG2 cells in a monolayer and three-dimensional high density cell culture by use of a matrigrid® (pages 2504–2512)

      Frank Weise, Uta Fernekorn, Jörg Hampl, Maren Klett and Andreas Schober

      Version of Record online: 22 APR 2013 | DOI: 10.1002/bit.24912

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      The image shows a cross-section of one microcavity of a MatriGrid® with HepG2 cells. It could be shown that the cells in a three-dimensional cell culture consume less oxygen relative to a monolayer cell culture and that the actively perfused three-dimensional cell culture in the MatriGrid® has a similar growth rate and vitality as the monolayer.

    21. Engineering Science of Biological Systems

      Oxygen control of intracellular distribution of mitochondria in muscle fibers (pages 2513–2524)

      B. Pathi, S. T. Kinsey and B. R. Locke

      Version of Record online: 30 APR 2013 | DOI: 10.1002/bit.24918

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      Mitochondrial distribution patterns in muscle fibers for a given oxygen concentration at the capillaries are described. The different redistribution patterns of mitochondria result from different values of the model parameter that reflects mitochondrial death as a result of oxygen concentration. Lower values of this parameter reflect higher sensitivity of mitochondrial death to oxygen and cause more redistribution to the edges.

    22. Systems Biotechnology

      Regiospecific modifications of naringenin for astragalin production in Escherichia coli (pages 2525–2535)

      Sailesh Malla, Ramesh Prasad Pandey, Byung-Gee Kim and Jae Kyung Sohng

      Version of Record online: 22 APR 2013 | DOI: 10.1002/bit.24919

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      Metabolic engineering and overexpression studies were carried out to increase intracellular UDP-glucose pool and regiospecific modifications of naringenin into astragalin in E.coli. This study demonstrates that the fermentation of E. coli cell factories is an excellent and alternative approach to produce flavonoid glycosides.

    23. Tissue Engineering and Delivery Systems

      Model predicting impact of complexation with cyclodextrins on oral absorption (pages 2536–2547)

      Ece D. Gamsiz, Avinash G. Thombre, Imran Ahmed and Rebecca L. Carrier

      Version of Record online: 10 JUL 2013 | DOI: 10.1002/bit.24932

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      Quantitative prediction of the influence of complexation with CD on oral drug absorption is enabled through development of a model considering simultaneous dynamic processes in the intestinal environment.

    24. Suspension culture of hepatocyte-derived reporter cells in presence of albumin to form stable three-dimensional spheroids (pages 2548–2555)

      C. Andrew Weeks, Kristen Newman, Paul A. Turner, Brian Rodysill, Raymond D. Hickey, Scott L. Nyberg and Amol V. Janorkar

      Version of Record online: 2 MAY 2013 | DOI: 10.1002/bit.24899

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      H35 rat hepatoma cells were cultured in a 24-well TCPS plate placed atop an orbital shaker to create 3-dimensional spheroids. The authors used BSA, dissolved in the cell culture medium and/or coated on TCPS surfaces, to provide stability to the formed spheroids in suspension configuration. Results demonstrate that BSA coating led to uniform and well-defined spheroids with little spheroid settling. In BSA-coated suspension systems, spheroid size scaled with the amount of BSA dissolved in culture medium.

  6. Communication to the Editor

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlights
    5. Viewpoint
    6. Articles
    7. Communication to the Editor
    1. Cellular and Metabolic Engineering

      Optimization of amorphadiene synthesis in bacillus subtilis via transcriptional, translational, and media modulation (pages 2556–2561)

      Kang Zhou, Ruiyang Zou, Congqiang Zhang, Gregory Stephanopoulos and Heng-Phon Too

      Version of Record online: 7 APR 2013 | DOI: 10.1002/bit.24900

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      In this proof-of-concept study, production of amorphadiene (precursor to antimalaria drug artermisinin) was demonstrated in a GRAS (generally regarded as safe) Bacillus. Systematic optimization including transcriptional, translational and media modulation led to production of ∼20mg/L amorphadiene within 48h in shake flask scale. With the developed genetic tools and optimization strategies, this study laid the foundation for high level production of isoprenoid pharmaceuticals in the fast growing GRAS Bacillus.

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