Biotechnology and Bioengineering

Cover image for Vol. 110 Issue 11

November 2013

Volume 110, Issue 11

Pages C1–C1, fmi–fmv, vi–vi, 2803–3069

  1. Cover

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlight
    5. Articles
    6. Communications to the Editor
  2. Contents

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlight
    5. Articles
    6. Communications to the Editor
  3. Spotlight

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlight
    5. Articles
    6. Communications to the Editor
  4. Articles

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlight
    5. Articles
    6. Communications to the Editor
    1. Biocatalysis, Protein Engineering, and Nanobiotechnology

      Crystallographic analysis and structure-guided engineering of NADPH-dependent Ralstonia sp. Alcohol dehydrogenase toward NADH cosubstrate specificity (pages 2803–2814)

      Alexandra Lerchner, Alexander Jarasch, Winfried Meining, André Schiefner and Arne Skerra

      Version of Record online: 1 JUL 2013 | DOI: 10.1002/bit.24956

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      The crystal structure of the NADP+-dependent alcohol dehydrogenase from Ralstonia sp. (RasADH)– a promising biocatalyst for industrial biotechnology –was solved in its apo- as well as in its NADP+-bound state. Structural determinants of stereospecificity were elucidated by docking of the established model substrate (S)-1-phenylethanol into the active site of the RasADH·NADP+ complex. Preferred acceptance of the recycable cofactor NAD+ was achieved by introducing for amino acid substitutions: N15G/G37D/R38V/R39S. X-ray analysis was applied to reveal their structural role.

    2. Semi-rational engineering of cytochrome P450sca-2 in a hybrid system for enhanced catalytic activity: Insights into the important role of electron transfer (pages 2815–2825)

      Lina Ba, Pan Li, Hui Zhang, Yan Duan and Zhanglin Lin

      Version of Record online: 4 JUN 2013 | DOI: 10.1002/bit.24960

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      This work used iterative saturation mutagenesis (ISM) to engineer an industrially important enzyme P450sca-2. Five sites located in the substrate binding pocket, substrate access entrance, and presumed electron transfer interface were rationally targeted. The best mutant showed a whole cell biotransformation activity and an overall apparent kcat that was 7.1-fold and 10.0-fold that of the starting template, respectively. Enhanced electron transfer was found to be most critical in the improvement, underscoring its importance in a hybrid P450 system.

    3. Production of anti-cancer immunotoxins in algae: Ribosome inactivating proteins as fusion partners (pages 2826–2835)

      Miller Tran, Ryan E. Henry, David Siefker, Christina Van, Greg Newkirk, Julie Kim, Jack Bui and Stephen P. Mayfield

      Version of Record online: 25 JUN 2013 | DOI: 10.1002/bit.24966

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      Mayfield has demonstrated the unique ability of algae to accumulate complex immunotoxin proteins, that would normally be toxic to eukaryotic cells, by producing them in the chloroplast. Once purified, these proteins are able to target and inhibit cancer cell proliferation in a dose-dependent matter. This research demonstrates the potential of using algae to produce these next generation cancer therapies.

    4. Investigation of the porous structure of cellulosic substrates through confocal laser scanning microscopy (pages 2836–2845)

      Dong Yang, Jose M. Moran-Mirabal, Jean-Yves Parlange and Larry P. Walker

      Version of Record online: 4 JUN 2013 | DOI: 10.1002/bit.24958

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      Fluorescently labeled dextrans were used as probes to assess their diffusion into the porous structure of filter paper. Nonlinear least squares fitting of the datasets to the pore grouping diffusion model yielded estimations of the micro-pore diffusion coefficient that described the inherently porous structure of plant-derived cellulose. Modeling results show on average 75% of the accessible pore volume is available for fast diffusion without any significant pore hindrance.

    5. Production of non-proteinogenic amino acids from α-keto acid precursors with recombinant Corynebacterium glutamicum (pages 2846–2855)

      Ju-Yeon Kim, Young-A Lee, Christoph Wittmann and Jin-Byung Park

      Version of Record online: 4 JUN 2013 | DOI: 10.1002/bit.24962

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      The non-proteinogenic amino acids (i.e., 2-(3-hydroxy-1-adamantyl)-(2S)-amino ethanoic acid, L-tert-leucine) could be efficiently produced from the corresponding α-keto acids by recombinant C. glutamicum expressing a transaminase of Escherichia coli.

    6. Development of a periplasmic FRET screening method for protease inhibitory antibodies (pages 2856–2864)

      Dong Hyun Nam and Xin Ge

      Version of Record online: 6 JUN 2013 | DOI: 10.1002/bit.24964

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      This article presents a function-based inhibitory antibody screening method, which combines a simple periplasmic preparation and an ultra sensitive FRET assay. Inhibitory antibodies can be rapidly distinguished from non-inhibitory clones with satisfactory Z-factors, thus making the method suitable for high-throughput applications. IC50 values of inhibition can also be estimated semi-quantitatively without antibody purification. The authors expect that these developments will expedite the generation of highly selective biologics, inhibiting many proteases important to medical research and therapeutic development.

    7. High-throughput screening for transglutaminase activities using recombinant fluorescent proteins (pages 2865–2873)

      Jae-Hun Lee, Eunjung Song, Sun-Gu Lee and Byung-Gee Kim

      Version of Record online: 10 JUL 2013 | DOI: 10.1002/bit.24970

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      Microbial transglutaminases from Bacillus sp. (BTGs) can be over-expressed as soluble proteins in E.coli host. In this study, the quenching between two recombinant fluorescent proteins by BTG activity was confirmed. This method can be applied to screen not only the substrates of TGs but also TG mutants in E.coli.

    8. Biofuels and Environmental Biotechnology

      Directed evolution of a highly efficient cellobiose utilizing pathway in an industrial Saccharomyces cerevisiae strain (pages 2874–2881)

      Yongbo Yuan and Huimin Zhao

      Version of Record online: 27 JUN 2013 | DOI: 10.1002/bit.24946

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      A novel strategy combining directed evolution and promoter engineering for rapid and efficient multi-gene pathway optimization was developed in this study, which provides potential applications in balancing and increasing the flux through an engineered heterologous pathway in a target organism to achieve high yield and productivity. By using this strategy, an industrial Saccharomyces cerevisiae strain capable of efficiently utilizing cellobiose was obtained.

    9. Optimization of outdoor cultivation in flat panel airlift reactors for lipid production by Chlorella vulgaris (pages 2882–2893)

      Ronja Münkel, Ulrike Schmid-Staiger, Achim Werner and Thomas Hirth

      Version of Record online: 16 MAY 2013 | DOI: 10.1002/bit.24948

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      An outdoor pilot plant with three 30 L flat panel airlift (FPA) reactors was operated in Stuttgart, Germany to produce high concentrated microalgae biomass with a lipid content around 50%. The main focus was on optimizing carbon dioxide and light availability. By adjusting the inoculum concentration the lipid productivity could be improved up to 0.39 g L−1 d−1 for a batch process of twelve days.

    10. Application of high throughput pretreatment and co-hydrolysis system to thermochemical pretreatment. Part 2: Dilute alkali (pages 2894–2901)

      Hongjia Li, Xiadi Gao, Jaclyn D. DeMartini, Rajeev Kumar and Charles E. Wyman

      Version of Record online: 10 JUN 2013 | DOI: 10.1002/bit.24951

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      Following achievements of hydrothermal and dilute acid conditions in high throughput pretreatment and enzymatic hydrolysis (HTPH) systems, a dilute alkali HTPH system was also successfully applied to offer additional insights for better understanding the roles of hemicellulose and lignin, respectively, in defining biomass recalcitrance. By employing the single-step neutralization and buffering concept, different biomass samples after various sodium hydroxide pretreatment conditions were directly conducted for co-hydrolysis. Sugar yield results demonstrated the HTPH system a clearly discernible screening tool for alkali conditions.

    11. Bioprocess Engineering and Supporting Technologies

      The changing dielectric properties of CHO cells can be used to determine early apoptotic events in a bioprocess (pages 2902–2914)

      Katrin Braasch, Marija Nikolic-Jaric, Tim Cabel, Elham Salimi, Greg E. Bridges, Doug J. Thomson and Michael Butler

      Version of Record online: 3 JUL 2013 | DOI: 10.1002/bit.24976

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      A prototype dielectrophoretic (DEP) cytometer has been developed to analyze individual CHO cells subjected to a radiofrequency actuator in a narrow bore capillary. Cell samples during the course of a bioreactor run show distinct shifts in the dielectric properties corresponding to loss of cell viability. Discrete cell sub-populations can be identified during apoptosis and can be correlated with alternative measurements by fluorescent markers and a cell population-based capacitance probe.

    12. Bioseparations and Downstream Processing

      Feasibility study of semi-selective protein precipitation with salt-tolerant copolymers for industrial purification of therapeutic antibodies (pages 2915–2927)

      Florian Capito, Johann Bauer, Almut Rapp, Christian Schröter, Harald Kolmar and Bernd Stanislawski

      Version of Record online: 20 JUN 2013 | DOI: 10.1002/bit.24950

      Copolymer- mediated selective precipitation was elucidated for an antibody capturing process. Compared to standard purification processes, this emerging technique is more cost-effective when using high titer mAb processes and easier to scale up.

    13. You have full text access to this OnlineOpen article
      Development of a novel and efficient cell culture flocculation process using a stimulus responsive polymer to streamline antibody purification processes (pages 2928–2937)

      Yun (Kenneth) Kang, James Hamzik, Michael Felo, Bo Qi, Julia Lee, Stanley Ng, Gregory Liebisch, Behnam Shanehsaz, Nripen Singh, Kris Persaud, Dale L. Ludwig and Paul Balderes

      Version of Record online: 29 JUN 2013 | DOI: 10.1002/bit.24969

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      A flocculation process using a stimulus responsive polymer enables a robust alternative cell culture harvest step which may debottleneck downstream processes, particularly for challenging monoclonal antibodies. Acceptable process yield and efficient clearance of HCP, host DNA, HMW, and residual polymer were demonstrated using five model antibodies.

    14. Cellular and Metabolic Engineering

      Strain optimization for efficient isobutanol production using Corynebacterium glutamicum under oxygen deprivation (pages 2938–2948)

      Shogo Yamamoto, Masako Suda, Satoko Niimi, Masayuki Inui and Hideaki Yukawa

      Version of Record online: 6 JUN 2013 | DOI: 10.1002/bit.24961

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      In this study, the authors describe metabolic engineering of Corynebacterium glutamicum, and optimization of reaction process for improved production of isobutanol under oxygen deprivation. Recombinant C. glutamicum using adhP gene with high isobutanol dehydrogenase activity produced 343 mM of isobutanol, the highest figure reported to date. Furthermore, restriction of by-product formation, redox balance improvement, and using oleyl alcohol to continuously extract the isobutanol, improved volumetric isobutanol productivity to 981 mM and the yield to 78.1%.

    15. Non-invasive in situ monitoring and quantification of TOL plasmid segregational loss within Pseudomonas putida biofilms (pages 2949–2958)

      Hongyan Ma, Kristy N. Katzenmeyer and James D. Bryers

      Version of Record online: 23 MAY 2013 | DOI: 10.1002/bit.24953

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      In situ estimation of plasmid segregational loss in a developing biofilm is shown. A. Spatial distribution of yellow fluorescent host cells (P. putida TUM-PP12 (miniTn5Putdsred) harboring the TOL plasmid pWWO::gfpmut3b)) and red fluorescent segregants (P. putida TUM-PP12 (miniTn5Putdsred)). The stack mage was collected on Day 4 of continuous biofilm formation. Arrows indicate the red fluorescent egregants embedded in microcolonies. Bar size: 20 µm. B. Distribution of both plasmid-bearing () and plasmid-free () cells at the different biofilm depths.

    16. Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum (pages 2959–2969)

      Sung Sun Yim, Seul Ji An, Misuk Kang, Jinho Lee and Ki Jun Jeong

      Version of Record online: 3 JUN 2013 | DOI: 10.1002/bit.24954

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      From the fully synthetic promoter library, the promoters available in Corynebacterium glutamicum were successfully isolated by FACS-based high-throughput screening. Through the extensive analysis of sequences and mRNA transcripts, the isolated promoters with various strengths were characterized. With two model proteins (antibody fragment and endoxylanase), the potential of the isolated proteins were also verified in flask cultivation as well as fed-batch fermentation. The screening strategy and the isolated promoters will contribute to the engineering of C. glutamicum host.

    17. CHO cell line specific prediction and control of recombinant monoclonal antibody N-glycosylation (pages 2970–2983)

      Rhian K. Grainger and David C. James

      Version of Record online: 6 JUN 2013 | DOI: 10.1002/bit.24959

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      Grainger and James demonstrate that it is possible to predict and control CHO cell N-glycan processing of a recombinant MAb using a combination of statistical modelling and comparative measurement of cell surface glycans using fluorescent lectins. Using design of experiments response surface modelling (DoE-RSM) it was possible to adjust relative concentrations of known metabolic effectors. MAb β1,4-galactosylation was improved >100 % compared to control non-supplemented cultures, and this was significantly correlated with cell surface galactosylation, enabling facile indirect prediction of N-glycan processing.

    18. Exogenous 1,4-butyrolactone stimulates A-factor-like cascade and validamycin biosynthesis in Streptomyces hygroscopicus 5008 (pages 2984–2993)

      Gao-Yi Tan, Linquan Bai and Jian-Jiang Zhong

      Version of Record online: 2 AUG 2013 | DOI: 10.1002/bit.24965

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      Validamycin A (VAL-A), produced by Streptomyces hygroscopicus, is the most widely used rice sheath blight controller. 1,4-Butyrolactone, as an easily accessible and cheap analogue of γ-butyrolactone, a family of important signaling molecules in regulation of secondary metabolism in Streptomyces, was applied to the VAL-A fermentation and found to enhance its production by stimulating A-factor-like cascade. The work provides a simple and efficient fermentation strategy and demonstrates the regulatory mechanism of A-factor-like cascade on VAL-A biosynthesis.

    19. Engineering Science of Biological Systems

      Enzymatic activity preservation and protection through entrapment within degradable hydrogels (pages 2994–3002)

      Angela M. Mariani, Mary E. Natoli and Peter Kofinas

      Version of Record online: 2 AUG 2013 | DOI: 10.1002/bit.24971

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      This work investigates the use of an abiotic entrapment method with potential to suspend enzyme activity and then enable reactivation on-demand—at site and time dictated by the user through degradation of the network crosslinks. This method fulfills the need for an easy, reliable, and repeatable enzymatic immobilization technique for potential use in production of high purity biomolecules, the natural degradation of molecular hazards, or for future biosensor applications.

    20. Synthetic Biology

      Peptide-based communication system enables Escherichia coli to Bacillus megaterium interspecies signaling (pages 3003–3012)

      Nicholas Marchand and Cynthia H. Collins

      Version of Record online: 5 JUL 2013 | DOI: 10.1002/bit.24975

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      Synthetic interspecies communication between Gram-negative Escherichia coli and Gram-positive Bacillus megaterium was established using components from the Staphylococcus aureus agr quorum-sensing system. Here, Marchand and Collins show that this communication system, which uses a novel peptide signal molecule, allows E. coli to control B. megaterium gene expression during co-culture.

    21. Systems Biotechnology

      Systems-wide analysis and engineering of metabolic pathway fluxes in bio-succinate producing Basfia succiniciproducens (pages 3013–3023)

      Judith Becker, Jasper Reinefeld, René Stellmacher, Rudolf Schäfer, Anna Lange, Hanna Meyer, Michael Lalk, Oskar Zelder, Gregory von Abendroth, Hartwig Schröder, Stefan Haefner and Christoph Wittmann

      Version of Record online: 9 JUL 2013 | DOI: 10.1002/bit.24963

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      Basfia succiniciproducens, a succinate producing rumen isolate, was investigated by 13C metabolic flux analysis. Systems-wide pathway fluxes suggested pyruvate-formate lyase and lactate dehydrogenase as the most promising targets towards strain improvement. Subsequent deletion of the encoding genes boosted succinate production by 45% up to a yield of 1.08 mol mol−1.

    22. Tissue Engineering and Delivery Systems

      Improving efficiency of human pluripotent stem cell differentiation platforms using an integrated experimental and computational approach (pages 3024–3037)

      Joshua A. Selekman, Amritava Das, Nicholas J. Grundl and Sean P. Palecek

      Version of Record online: 9 JUL 2013 | DOI: 10.1002/bit.24968

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      Quantitative analysis of hPSC differentiation systems to identify bottlenecks to differentiation efficiency is described. Selekman and coworkers have compartmentalized an epithelial hPSC differentiation system by identifying stable cell states and distinguishing these states using different marker proteins. By fitting an ODE-based model to data representing dynamics of these cell states, the authors have estimated rates of various cell fate decisions and determined which decisions are potentially limiting to the differentiation process.

    23. Digital microfabrication of user-defined 3D microstructures in cell-laden hydrogels (pages 3038–3047)

      Pranav Soman, Peter H. Chung, A. Ping Zhang and Shaochen Chen

      Version of Record online: 3 JUN 2013 | DOI: 10.1002/bit.24957

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      Current tissue engineering strategies show limited success in rapidly fabricating 3D microenvironments featuring both complex topography and encapsulated cells. The authors present a novel microfabrication approach for constructing cell-laden biomaterial hydrogels that (1) provide complex user-defined 3D geometries, (2) allow for consistent 3D distribution of encapsulated cells, (3) support cell viability and proliferation, (4) feature dynamic, large-scale mechanical cell-scaffold interactions, and (5) yield cell behavior and morphology that contrast with outcomes from traditional cell seeding.

    24. Dorsal and ventral stimuli in sandwich-like microenvironments. Effect on cell differentiation (pages 3048–3058)

      José Ballester-Beltrán, Myriam Lebourg and Manuel Salmerón-Sánchez

      Version of Record online: 27 JUN 2013 | DOI: 10.1002/bit.24972

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      Dorsal stimulation of C2C12 myoblasts using sandwich-like microenvironments enhances cell differentiation. Dorsal integrin-mediated adhesion within sandwich culture triggers intracellular signal cascades and increases myoblast differentiation. Sandwich-like models are proposed as a versatile tool to study cell behavior in a quasi-3D environment under well-controlled conditions.

  5. Communications to the Editor

    1. Top of page
    2. Cover
    3. Contents
    4. Spotlight
    5. Articles
    6. Communications to the Editor
    1. Biofuels and Environmental Biotechnology

      Spatial distribution of bacterial communities on volumetric and planar anodes in single-chamber air-cathode microbial fuel cells (pages 3059–3062)

      Ignacio T. Vargas, Istvan U. Albert and John M. Regan

      Version of Record online: 29 MAY 2013 | DOI: 10.1002/bit.24949

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      Pyrosequencing was used to characterize bacterial communities in air-cathode microbial fuel cells across a volumetric (graphite fiber brush) and a planar (carbon cloth) anode, where different physical and chemical gradients would be expected associated with the distance between anode location and the air-cathode. Geobacter spp. and other dominant bacterial groups were homogenously distributed across both planar and volumetric anodes. This result lends support to previous community analysis interpretations based on a single biofilm sampling location in these systems.

    2. Bioseparations and Downstream Processing

      Design, synthesis, and assessment of a de novo affinity adsorbent for the purification of recombinant human erythropoietin (pages 3063–3069)

      Graziella El Khoury, Yian Wang, Di Wang, Shaleem I. Jacob and Christopher R. Lowe

      Version of Record online: 16 MAY 2013 | DOI: 10.1002/bit.24943

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      This work describes the assessment of a synthetic affinity ligand (A9C10I8) for human recombinant erythropoietin (rHuEPO) based on the multicomponent Ugi reaction. The design of the ligand, synthesized on aldehyde-functionalized agarose beads, was based on the analysis of the X-ray crystallographic structure of the complex between the EPO and its cell-surface receptor (EPObp)2. This study demonstrates the potential of Ugi scaffolds to generate affinity adsorbents for the future development of cost-effective purification protocols for rHuEPO.

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