Journal Cover Biotechnology and Bioengineering
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   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0006-3592 - ISSN (Online) 1097-0290
   Published by John Wiley and Sons Homepage  [1592 journals]
  • Peptide ligand-mediated endocytosis of nanoparticles to cancer cells: cell
           receptor-binding- vs. cell membrane-penetrating peptides
    • Authors: Eunji Jo; June Seok Heo, Ja-Yun Lim, Bo-Ram Lee, Chul Joo Yoon, Jinkwan Kim, Jeewon Lee
      Abstract: The endocytosis-mediating performances of two types of peptide ligands, cell receptor binding peptide (CRBP) and cell membrane penetrating peptide (CMPP), were analyzed and compared using a common carrier of peptide ligands-human ferritin heavy chain (hFTH) nanoparticle. 24 copies of a CMPP(human immunodeficiency virus-derived TAT peptide) and/or a CRBP (peptide ligand with strong and specific affinity for either human integrin(αvβ3) or epidermal growth factor receptor I (EGFR) that is overexpressed on various cancer cells) were genetically presented on the surface of each hFTH nanopariticle. The quantitative level of endocytosis and intracellular localization of fluorescence dye-labeled CRBP- and CMPP-presenting nanoparticles were estimated in the in vitro cultures of integrin- and EGFR-overexpressing cancer and human dermal fibroblast cells(control). From the cancer cell cultures treated with the CMPP- and CRBP-presenting nanoparticles, it was notable that CRBPs resulted in quantitatively higher level of endocytosis than CMPP (TAT) and successfully transported the nanoparticles to the cytosol of cancer cells depending on concentration and treatment period of time, whereas TAT-mediated endocytosis localized most of the nanoparticles within endosomal vesicles under the same conditions. These novel findings provide highly useful informations to many researchers both in academia and in industry who are interested in developing anticancer drug delivery systems/carriers. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-20T07:55:46.736584-05:
      DOI: 10.1002/bit.26575
  • Harnessing a Methane-Fueled, Sediment-Free Mixed Microbial Community for
           Utilization of Distributed Sources of Natural Gas
    • Authors: Jeffrey J. Marlow; Amit Kumar, Brandon Enalls, Linda M. Reynard, Noreen Tuross, Gregory Stephanopoulos, Peter Girguis
      Abstract: Harnessing the metabolic potential of uncultured microbial communities is a compelling opportunity for the biotechnology industry, an approach that would vastly expand the portfolio of usable feedstocks. Methane is particularly promising because it is abundant and energy-rich, yet the most efficient methane-activating metabolic pathways involve mixed communities of anaerobic methanotrophic archaea and sulfate reducing bacteria. These communities oxidize methane at high catabolic efficiency and produce chemically reduced by-products at a comparable rate and in near-stoichiometric proportion to methane consumption. These reduced compounds can be used for feedstock and downstream chemical production, and at the production rates observed in situ they are an appealing, cost-effective prospect. Notably, the microbial constituents responsible for this bioconversion are most prominent in select deep-sea sediments, and while they can be kept active at surface pressures, they have not yet been cultured in the lab. In an industrial capacity, deep-sea sediments could be periodically recovered and replenished, but the associated technical challenges and substantial costs make this an untenable approach for full-scale operations.In this study, we present a novel method for incorporating methanotrophic communities into bioindustrial processes through abstraction onto low mass, easily transportable carbon cloth artificial substrates. Using Gulf of Mexico methane seep sediment as inoculum, optimal physicochemical parameters were established for methane-oxidizing, sulfide-generating mesocosm incubations. Metabolic activity required>∼40% seawater salinity, peaking at 100% salinity and 35 °C. Microbial communities were successfully transferred to a carbon cloth substrate, and rates of methane-dependent sulfide production increased more than three-fold per unit volume. Phylogenetic analyses indicated that carbon cloth-based communities were substantially streamlined and were dominated by Desulfotomaculum geothermicum. Fluorescence in situ hybridization microscopy with carbon cloth fibers revealed a novel spatial arrangement of anaerobic methanotrophs and sulfate reducing bacteria suggestive of an electronic coupling enabled by the artificial substrate. This system 1) enables a more targeted manipulation of methane-activating microbial communities using a low-mass and sediment-free substrate, 2) holds promise for the simultaneous consumption of a strong greenhouse gas and the generation of usable downstream products, and 3) furthers the broader adoption of uncultured, mixed microbial communities for biotechnological use. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-20T07:50:36.853974-05:
      DOI: 10.1002/bit.26576
  • Enhancing cytochrome P450-mediated non-natural cyclopropanation by
           mutation of a conserved second-shell residue
    • Authors: Joshua G. Gober; Amy E. Rydeen, Timothy D. Schwochert, Evan J. Gibson-O'Grady, Eric M. Brustad
      Abstract: Engineered cytochrome P450s are emerging as powerful synthetic tools due to their ability catalyze non-native metallocarbenoid and –nitrenoid insertion reactions. P450-mediated cyclopropanation has garnered particular interest due to the high selectivity demonstrated by engineered scaffolds and their application towards the synthesis of therapeutic agents. We previously reported that mutation of a conserved, first-shell heme-ligating Cys to Ser led to significant improvements in cyclopropanation activity in a model enzyme, P450BM3h. Here, we demonstrate that mutation of a ubiquitously conserved second-shell Phe (F393) to His or Ala, provides complementary increases in the P450 heme reduction potential and conversion to cyclopropanation products when compared to first-shell CS mutations. Furthermore, we show that these mutations confer improved non-natural catalysis in 4 diverse P450 scaffolds. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-20T04:56:10.874476-05:
      DOI: 10.1002/bit.26571
  • Engineered CRISPR/Cas9 System for Multiplex Genome Engineering of
           Polyploid Industrial Yeast Strains
    • Authors: Jiazhang Lian; Zehua Bao, Sumeng Hu, Huimin Zhao
      Abstract: The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. However, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to construct xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-20T04:56:08.771576-05:
      DOI: 10.1002/bit.26569
  • A perfusion chamber for monitoring transepithelial NaCl transport in an in
           vitro model of the renal tubule
    • Authors: Jose Yeste; Laura Martínez-Gimeno, Xavi Illa, Pablo Laborda, Anton Guimerà, Juan P. Sánchez-Marín, Rosa Villa, Ignacio Giménez
      Abstract: Transepithelial electrical measurements in the renal tubule have provided a better understanding of how kidney regulates electrolyte and water homeostasis through the reabsorption of molecules and ions (e.g., H2O and NaCl). While experiments and measurement techniques using native tissue are difficult to prepare and to reproduce, cell cultures conducted largely with the Ussing chamber lack the effect of fluid shear stress which is a key physiological stimulus in the renal tubule. To overcome these limitations, we present a modular perfusion chamber for long-term culture of renal epithelial cells under flow that allows the continuous and simultaneous monitoring of both transepithelial electrical parameters and transepithelial NaCl transport. The latter is obtained from electrical conductivity measurements since Na+ and Cl- are the ions that contribute most to the electrical conductivity of a standard physiological solution. The system was validated with epithelial monolayers of raTAL and NRK-52E cells that were characterized electrophysiologically for five days under different flow conditions (i.e., apical perfusion, basal, or both). In addition, apical to basal chemical gradients of NaCl (140/70 and 70/140 mM) were imposed in order to demonstrate the feasibility of this methodology for quantifying and monitoring in real time the transepithelial reabsorption of NaCl, which is a primary function of the renal tubule. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-20T04:55:36.834924-05:
      DOI: 10.1002/bit.26574
    • Authors: Mia Mae Kiamco; Abdelrhman Mohamed, Patrick Reardon, Carrie Marean-Reardon, Wrya Moh Aframehr, Douglas R. Call, Haluk Beyenal, Ryan Renslow
      Abstract: Biofilms alter their metabolism in response to environmental stress. This study explores the effect of a hyperosmotic agent–antibiotic treatment on the metabolism of Staphylococcus aureus biofilms through the use of nuclear magnetic resonance (NMR) techniques. To determine the metabolic activity of S. aureus, we quantified the concentrations of metabolites in spent medium using high-resolution NMR spectroscopy. Biofilm porosity, thickness, biovolume, and relative diffusion coefficient depth profiles were obtained using NMR microimaging. Dissolved oxygen concentration was measured to determine the availability of oxygen within the biofilm. Under vancomycin-only treatment, the biofilm communities switched to fermentation under anaerobic condition, as evidenced by high concentrations of formate (7.4 ± 2.7 mM), acetate (13.1 ± 0.9 mM), and lactate (3.0 ± 0.8 mM), and there was no detectable dissolved oxygen in the biofilm. Anaerobic conditions such as fermentation signify that biofilm is combating antibiotic stress by developing resistance. In addition, we observed the highest consumption of pyruvate (0.19 mM remaining from an initial 40 mM concentration), the sole carbon source, under the vancomycin-only treatment. On the other hand, relative effective diffusion coefficients increased from 0.73 ± 0.08 to 0.88 ± 0.08 under vancomycin-only treatment but decreased from 0.71 ± 0.04 to 0.60 ± 0.07 under maltodextrin-only and from 0.73 ± 0.06 to 0.56 ± 0.08 under combined treatments. There was an increase in biovolume, from 2.5 ± 1 mm3 to 7 ± 1 mm3, under the vancomycin-only treatment, while the maltodextrin-only and combined treatments showed no significant change in biovolume over time. This indicated that physical biofilm growth was halted during maltodextrin-only and combined treatments. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-20T04:55:31.315748-05:
      DOI: 10.1002/bit.26572
  • Fluorescent Nanodiamond-bacteriophage Conjugates Maintain Host Specificity
    • Authors: Jimmy T. Trinh; Masfer H. Alkahtani, Isaac Rampersaud, Arfaan Rampersaud, Marlan Scully, Ryland F. Young, Philip Hemmer, Lanying Zeng
      Abstract: Rapid identification of specific bacterial strains within clinical, environmental, and food samples can facilitate the prevention and treatment of disease. Fluorescent nanodiamonds (FNDs) are being developed as biomarkers in biology and medicine, due to their excellent imaging properties, ability to accept surface modifications, and lack of toxicity. Bacteriophages, the viruses of bacteria, can have exquisite specificity for certain hosts. We propose to exploit the properties of FNDs and phages to develop phages conjugated with FNDs as long-lived fluorescent diagnostic reagents. In this study, we develop a simple procedure to create such fluorescent probes by functionalizing the FNDs and phages with streptavidin and biotin, respectively. We find that the FND-phage conjugates retain the favorable characteristics of the individual components and can discern their proper host within a mixture. This technology may be further explored using different phage/bacteria systems, different FND color centers and alternate chemical labeling schemes for additional means of bacterial identification and new single-cell/virus studies. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-20T04:55:23.570108-05:
      DOI: 10.1002/bit.26573
  • RNAi Assisted Genome Evolution Unveils Yeast Mutants with Improved Xylose
    • Authors: Mohammad HamediRad; Jiazhang Lian, Hejun Li, Huimin Zhao
      Abstract: Xylose is a major component of lignocellulosic biomass, one of the most abundant feedstocks for biofuel production. Therefore, efficient and rapid conversion of xylose to ethanol is crucial in the viability of lignocellulosic biofuel plants. In this study, RNAi Assisted Genome Evolution (RAGE) was used to improve the xylose utilization rate in SR8, one of the most efficient publicly available xylose utilizing Saccharomyces cerevisiae strains. To identify gene targets for further improvement, we created a genome-scale library consisting of both genetic over-expression and down-regulation mutations in SR8. Followed by screening in media containing xylose as the sole carbon source, yeast mutants with 29% faster xylose utilization and 45% higher ethanol productivity were obtained relative to the parent strain. Two known and two new effector genes were identified in these mutant strains. Notably, down-regulation of CDC11, an essential gene, resulted in faster xylose utilization, and this gene target cannot be identified in genetic knock-out screens. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-20T04:50:24.268611-05:
      DOI: 10.1002/bit.26570
  • Application of adaptive laboratory evolution to overcome a flux limitation
           in an Escherichia coli production strain
    • Authors: Kento Tokuyama; Yoshihiro Toya, Takaaki Horinouchi, Chikara Furusawa, Fumio Matsuda, Hiroshi Shimizu
      Abstract: Gene deletion strategies using flux balance analysis (FBA) have improved the growth-coupled production of various compounds. However, the productivities were often below the expectation because the cells failed to adapt to these genetic perturbations. Here, we demonstrate the productivity of the succinate of the designed gene deletion strain was improved by adaptive laboratory evolution (ALE). Although FBA predicted deletions of adhE-pykAF-gldA-pflB lead to produce succinate from glycerol with a yield of 0.45 C-mol/C-mol, the knockout mutant did not produce only 0.08 C-mol/Cmol, experimentally. After the ALE experiments, the highest succinate yield of an evolved strain reached to the expected value. Genome sequencing analysis revealed all evolved strains possessed novel mutations in ppc of I829S or R849S. In vitro enzymatic assay and metabolic profiling analysis revealed that these mutations desensitizing an allosteric inhibition by L-aspartate and improved the flux through Ppc, while the activity of Ppc in the unevolved strain was tightly regulated by L-aspartate. These result demonstrated that the evolved strains achieved the improvement of succinate production by expanding the flux space of Ppc, realizing the predicted metabolic state by FBA. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-19T07:25:47.860182-05:
      DOI: 10.1002/bit.26568
  • Combination Drug Delivery via Multilamellar Vesicles Enables Targeting of
           Tumor Cells and Tumor Vasculature
    • Authors: Yarong Liu; Yu Jeong Kim, Natnaree Siriwon, Jennifer A. Rohrs, Zhiqiang Yu, Pin Wang
      Abstract: Blood vessel development is critical for the continued growth and progression of solid tumors and, therefore, makes an attractive target for improving cancer therapy. Indeed, vascular-targeted therapies have been extensively explored but they have shown minimal efficacy as monotherapies. Combretastatin A4 (CA-4) is a tubulin-binding vascular disrupting agent that selectively targets the established tumor endothelium, causing rapid vascular beak down. Despite its potent anticancer potential, the drug has dose-limiting side effects, particularly in the form of cardiovascular toxicity. Furthermore, its poor aqueous solubility and the resulting limited bioavailability hinder its antitumor activity in the clinic. To improve the therapeutic efficacy of CA-4, we investigated its application as a combination therapy with doxorubicin (Dox) in a tumor vasculature targeted delivery vehicle: peptide-modified cross-linked multilamellar liposomal vesicles (cMLVs). In vitro cell culture studies showed that a tumor vasculature-targeting peptide, RIF7, could facilitate higher cellular uptake of drug-loaded cMLVs and consequently enhance the antitumor efficacy in both drug resistant B16 mouse melanoma and human MDA-MB-231 breast cancer cells. In vivo, upon intravenous injection, targeted cMLVs could efficiently deliver both Dox and CA-4 to significantly slow tumor growth through the specific interaction of the targeting peptide with its receptor on the surface of tumor vasculature. This study demonstrates the potential of our novel targeted combination therapy delivery vehicle to improve the outcome of cancer treatment. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-19T07:20:30.38191-05:0
      DOI: 10.1002/bit.26566
  • Antibody Glycoengineering Strategies in Mammalian Cells
    • Authors: Qiong Wang; Cheng-Yu Chung, Sandra Chough, Michael J. Betenbaugh
      Abstract: As a key parameter impacting functional and structural heterogeneity, protein glycosylation is a critical quality attribute for antibody biotherapeutic manufacturing. The glycan patterns on recombinant antibodies, particularly on the conserved fragment crystallizable (Fc) region, can have significant effects on an antibody's functional activities including clearance rate, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and anti-inflammatory activity. In this review, we examined specific glycan attachments (fucosylation, sialylation, galactosylation, oligo-mannose and bisecting glycans) and their importance to antibody properties. Next, we summarized the recent and current achievements on controlling antibody glycoforms in Chinese hamster ovary (CHO) and other mammalian cells through multiple strategies including genetic engineering, protein engineering, media modification and other emerging technologies. Further, the impact of one carbohydrate modification on other glycan structures is also described. Finally, approaches to generate desirable homogenous glycan profiles on antibodies are also detailed. By applying multiple complementary intracellular and extracellular strategies, biotechnologists are well on their ways to precisely tuning antibody glycoforms emerging from bioreactors in the coming decades. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-19T07:20:23.704873-05:
      DOI: 10.1002/bit.26567
  • Rapid eradication of bacterial phytopathogens by atmospheric pressure glow
           discharge generated in contact with a flowing liquid cathode
    • Authors: Agata Motyka; Anna Dzimitrowicz, Piotr Jamroz, Ewa Lojkowska, Wojciech Sledz, Pawel Pohl
      Abstract: Diseases caused by phytopathogenic bacteria are responsible for significant economic losses, and these bacteria spread through diverse pathways including waterways and industrial wastes. It is therefore of interest to develop potent methods for their eradication. Here, antibacterial properties of direct current atmospheric pressure glow discharge (dc-APGD) generated in contact with flowing bacterial suspensions were examined against five species of phytopathogens. Complete eradication of Clavibacter michiganensis subsp. sepedonicus, Dickeya solani, and Xanthomonas campestris pv. campestris from suspensions of OD600≈0.1 was observed, while there was at least 3.43 logarithmic reduction in population densities of Pectobacterium atrosepticum and Pectobacterium carotovorum subsp. carotovorum. Analysis of plasma-chemical parameters of the dc-APGD system revealed its high rotational temperatures of 2300 ± 100 K and 4200 ± 200 K, as measured from N2 and OH molecular bands, respectively, electron temperature of 6050 ± 400 K, vibrational temperature of 4000 ± 300 K, and high electron number density of 1.1 × 1015 cm−1. In addition, plasma treatment lead to formation of numerous reactive species and states in the treated liquid, including reactive nitrogen and oxygen species such as NOx, NH, H2O2, O2, O, and OH. Further examinations revealed that bactericidal activity of dc-APGD was primarily due to presence of these reactive species as well as to UVA, UVB, and UVC irradiation generated by the dc-APGD source. Plasma treatment also resulted in an increase in temperature (from 24.2 to 40.2 °C) and pH (from 6.0 to 10.8) of bacterial suspensions, although these changes had minor effects on cell viability. All results suggest that the newly developed dc-APGD-based system can be successfully implemented as a simple, rapid, efficient, and cost-effective disinfection method for liquids originating from different industrial and agricultural settings. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-19T07:05:55.655875-05:
      DOI: 10.1002/bit.26565
  • A coupled in vitro/in vivo approach for engineering a heterologous Type
           III PKS to enhance polyketide biosynthesis in Saccharomyces cerevisiae
    • Authors: Christopher R. Vickery; Javier Cardenas, Marianne. Bowman, Michael D. Burkart, Nancy A. Da Silva, Joseph P. Noel
      Abstract: Polyketides are attractive compounds for uses ranging from biorenewable chemical precursors to high-value therapeutics. In many cases, synthesis in a heterologous host is required to produce these compounds in industrially relevant quantities. The type III polyketide synthase 2-pyrone synthase (2-PS) from Gerbera hybrida was used for the production of triacetic acid lactone (TAL) in Saccharomyces cerevisiae. Initial in vitro characterization of 2-PS led to the identification of active site variants with improved kinetic properties relative to wildtype. Further in vivo evaluation in S. cerevisiae suggested certain 2-PS mutations altered enzyme stability during fermentation. In vivo experiments also revealed beneficial cysteine to serine mutations that were not initially explored due to their distance from the active site of 2-PS, leading to the design of additional 2-PS enzymes. While these variants showed varying catalytic efficiencies in vitro, they exhibited up to 2.5-fold increases in TAL production when expressed in S. cerevisiae. Coupling of the 2-PS variant [C35S,C372S] to an engineered S. cerevisiae strain led to over 10 g/L TAL at 38% of theoretical yield following fed-batch fermentation, the highest reported. Our studies demonstrate the success of a coupled in vitro/in vivo approach to engineering enzymes and provide insight on cysteine-rich enzymes and design principles towards their use in non-native microbial hosts. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-19T07:05:53.176548-05:
      DOI: 10.1002/bit.26564
  • Anodic Electro-Fermentation: Anaerobic production of L-Lysine by
           recombinant Corynebacterium glutamicum
    • Authors: Igor Vassilev; Gideon Gießelmann, Susanne K. Schwechheimer, Christoph Wittmann, Bernardino Virdis, Jens O. Krömer
      Abstract: Microbial electrochemical technologies (MET) are promising to drive metabolic processes for the production of chemicals of interest. They provide microorganisms with an electrode as an electron sink or an electron source to stabilize their redox and/or energy state. Here, we applied an anode as additional electron sink to enhance the anoxic metabolism of the industrial bacterium Corynebacterium glutamicum through an anodic electro-fermentation. In using ferricyanide as extracellular electron carrier, anaerobic growth was enabled and the feedback-deregulated mutant Corynebacterium glutamicum lysC further accumulated L-lysine. Under such oxidising conditions we achieved L-lysine titers of 2.9 mM at rates of 0.2 mmol/L/h. That titer is comparable to recently reported L-lysine concentrations achieved by anaerobic production under reductive conditions (cathodic electro-fermentation). However unlike other studies, our oxidative conditions allowed anaerobic cell growth, indicating an improved cellular energy supply during anodic electrofermentation. In that light, we propose anodic electro-fermentation as the right choice to support C. glutamicum stabilizing its redox and energy state and empower a stable anaerobic production of L-lysine. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-10T07:35:33.70152-05:0
      DOI: 10.1002/bit.26562
  • Butyrated ManNAc analog improves protein expression in Chinese hamster
           ovary cells
    • Authors: Bojiao Yin; Qiong Wang, Cheng-Yu Chung, Xiaozhi Ren, Rahul Bhattacharya, Kevin J. Yarema, Michael J. Betenbaugh
      Abstract: The chemical additive sodium butyrate (NaBu) has been applied in cell culture media as a direct and convenient method to increase the protein expression in Chinese hamster ovary (CHO) and other mammalian cells. In this study, we examined an alternative chemical additive, 1,3,4-O-Bu3ManNAc, for its effect on recombinant protein production in CHO. Supplementation with 1,3,4-O-Bu3ManNAc for two stable CHO cell lines, expressing human erythropoietin or IgG, enhanced protein expression for both products with negligible impact on cell growth, viability, glucose utilization, and lactate accumulation. In contrast, sodium butyrate treatment resulted in a ∼20% decrease in maximal viable cell density and ∼30% decrease in cell viability at the end of cell cultures compared to untreated or 1,3,4-O-Bu3ManNAc treated CHO cell lines for both products. While NaBu treatment enhanced product yields more than the 1,3,4-O-Bu3ManNAc treatment, the NaBu treated cells also exhibited higher levels of caspase 3 positive cells using microscopy analysis. Furthermore, the mRNA levels of 4 cell apoptosis genes (Cul2, BAK, BAX and BCL2L11) were up-regulated more in sodium butyrate treated wild-type, erythropoietin or IgG expressing CHO-K1 cell lines while most of the mRNA levels of apoptosis genes in 1,3,4-O-Bu3ManNAc treated cell lines remained equal or increased only slightly compared to the levels in untreated CHO cell lines. Finally, lectin blot analysis revealed that the 1,3,4-O-Bu3ManNAc-treated cells displayed higher relative sialylation levels on recombinant EPO, consistent with the effect of the ManNAc component of this additive, compared to control while NaBu treatment led to lower sialylation levels than control or 1,3,4-O-Bu3ManNAc-treatment. These findings demonstrate that 1,3,4-O-Bu3ManNAc has fewer negative effects on cell cytotoxicity and apoptosis, perhaps as a result of a more deliberate uptake and release of the butyrate compounds, while simultaneously increasing the expression of multiple recombinant proteins and improving the glycosylation characteristics when applied at comparable molarity levels to NaBu. Thus, 1,3,4-O-Bu3ManNAc represents a highly promising media additive alternative in cell culture for improving protein yields without sacrificing cell mass and product quality in future bioproduction processes. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-10T07:35:29.090515-05:
      DOI: 10.1002/bit.26560
  • Differentiation of 3D-shape-controlled mouse neural stem cell to neural
           tissues in closed agarose microchambers
    • Authors: Yuki Matsushiro; Midori Kato-Negishi, Hiroaki Onoe
      Abstract: This paper describes three-dimensional (3D) tissue shape control of mouse neural stem cell (mNSC) micro tissues by using closed agarose microchambers for effective differentiation induction of neurons in vitro. Our agarose microchambers, made by micromolding, can be sealed with an agarose sheet to form the mNSC tissues along the shape of microchambers. We constructed lane-shaped mNSC tissues with different width (∼60–210 µm) and thickness (∼25–95 µm) dimensions and induced differentiation to neurons with differentiation medium. We found that in thick tissues (thickness:>60 µm), distribution of differentiated neurons was not uniform, whereas in thin tissues (thickness: ∼30 µm), differentiated neurons were uniformly distributed with high differentiation efficiency. Our system to construct in vitro 3D neural tissues having uniformly distributed neurons at high differentiation ratio, could become an effective tool for drug screening using 3D neural tissues and 3D mNSC tissues under differentiation induction. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-10T07:35:26.913961-05:
      DOI: 10.1002/bit.26559
  • Highly sensitive detection of mutations in CHO cell recombinant DNA using
           multi-parallel single molecule real-time DNA sequencing
    • Authors: Joseph F. Cartwright; Karin Anderson, Joseph Longworth, Philip Lobb, David C. James
      Abstract: High-fidelity replication of biologic-encoding recombinant DNA sequences by engineered mammalian cell cultures is an essential pre-requisite for the development of stable cell lines for the production of biotherapeutics. However, immortalized mammalian cells characteristically exhibit an increased point mutation frequency compared to mammalian cells in vivo, both across their genomes and at specific loci (hotspots). Thus unforeseen mutations in recombinant DNA sequences can arise and be maintained within producer cell populations. These may affect both the stability of recombinant gene expression and give rise to protein sequence variants with variable bioactivity and immunogenicity. Rigorous quantitative assessment of recombinant DNA integrity should therefore form part of the cell line development process and be an essential quality assurance metric for instances where synthetic/multi-component assemblies are utilized to engineer mammalian cells, such as the assessment of recombinant DNA fidelity or the mutability of single-site integration target loci.Based on Pacific Biosciences single molecule real-time (SMRT™) circular consensus sequencing (CCS) technology we developed a rDNA sequence analysis tool to process the multi-parallel sequencing of ∼40,000 single recombinant DNA molecules. After statistical filtering of raw sequencing data, we show that this analytical method is capable of detecting single point mutations in rDNA to a minimum single mutation frequency of 0.0042% (
      PubDate: 2018-02-10T07:35:23.565973-05:
      DOI: 10.1002/bit.26561
  • Proteomics in biomanufacturing control: Protein dynamics of CHO-K1 cells
           and conditioned media during apoptosis and necrosis
    • Authors: Simone Albrecht; Christian Kaisermayer, Clair Gallagher, Amy Farrell, Anna Lindeberg, Jonathan Bones
      Abstract: Cell viability has a critical impact on product quantity and quality during the biomanufacturing of therapeutic proteins. An advanced understanding of changes in the cellular and conditioned media proteomes upon cell stress and death is therefore needed for improved bioprocess control. Here, a high pH / low pH reversed phase data independent 2D-LC-MSE discovery proteomics platform was applied to study the cellular and conditioned media proteomes of CHO-K1 apoptosis and necrosis models where cell death was induced by staurosporine exposure or aeration shear in a benchtop bioreactor, respectively. Functional classification of gene ontology terms related to molecular functions, biological processes and cellular components revealed both cell death independent and specific features. In addition, label free quantitation using the Hi3 approach resulted in a comprehensive shortlist of 23 potential cell viability marker proteins with highest abundance and a significant increase in the conditioned media upon induction of cell death, including proteins related to cellular stress response, signal mediation, cytoskeletal organization, cell differentiation, cell interaction as well as metabolic and proteolytic enzymes which are interesting candidates for translating into targeted analysis platforms for monitoring bioprocessing response and increasing process control. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-10T07:30:26.048757-05:
      DOI: 10.1002/bit.26563
  • Photo-switchable Microbial Fuel-Cells
    • Authors: Orr Schlesinger; Rambabu Dandela, Ashok Bhagat, Michael M. Meijler, Lin Xia, Lital Alfonta
      Abstract: Regulation of Biosystems in a clean, simple and efficient way is important for the design of smart bio-interfaces and bioelecronic devices. Light as a non-invasive mean to control the activity of a protein enables spatial and temporal control far superior to other chemical and physical methods. The ability to regulate the activity of a catalytic enzyme in a biofuel-cell reduces the waste of resources and energy and turns the fuel-cell into a smart and more efficient device for power generation. Here we present a microbial-fuel-cell based on a surface displayed, photo-switchable alcohol dehydrogenase. The enzyme was modified near the active site using non-canonical amino acids and a small photo-reactive molecule, which enables reversible control over enzymatic activity. Depending on the modification site, the enzyme exhibits reversible behaviour upon irradiation with UV and visible light, in both biochemical and electrochemical assays. The change observed in power output in a microbial fuel cell utilizing the modified enzyme was almost 5-fold, between inactive and active states. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-07T07:01:24.540708-05:
      DOI: 10.1002/bit.26555
  • Purification, Characterization, and N-glycosylation of Recombinant
           Butyrylcholinesterase from Transgenic Rice Cell Suspension Cultures
    • Authors: Jasmine M. Corbin; Muchena J. Kailemia, C. Linn Cadieux, Salem Alkanaimsh, Kalimuthu Karuppanan, Raymond L. Rodriguez, Carlito B. Lebrilla, Douglas M. Cerasoli, Karen A. McDonald, Somen Nandi
      Abstract: Recombinant butyrylcholinesterase produced in a metabolically-regulated transgenic rice cell culture (rrBChE) was purified to produce a highly pure (95%), active form of enzyme. The developed downstream process uses common manufacturing-friendly operations including tangential flow filtration, anion-exchange chromatography, and affinity chromatography to obtain a process recovery of 42% active rrBChE. The purified rrBChE was then characterized to confirm its comparability to the native human form of the molecule (hBChE). The recombinant and native enzyme demonstrated comparable enzymatic behavior and had an identical amino acid sequence. However, rrBChE differs in that it contains plant-type complex N-glycans, including an α-1,3 linked core fucose and a β-1,2 xylose, and lacking a terminal sialic acid. Despite this difference, rrBChE is demonstrated to be an effective stoichiometric bioscavenger for 5 different organophosphorous nerve agents in vitro. Together, the efficient downstream processing scheme and functionality of rrBChE confirm its promise as a cost-effective alternative to hBChE for prophylactic and therapeutic use. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-07T06:56:25.639944-05:
      DOI: 10.1002/bit.26557
  • Improving methyl ketone production in E. coli by heterologous expression
           of NADH-dependent FabG
    • Authors: Ee-Been Goh; Yan Chen, Christopher J. Petzold, Jay D. Keasling, Harry R. Beller
      Abstract: We previously engineered E. coli to overproduce medium- to long-chain saturated and monounsaturated methyl ketones, which could potentially be applied as diesel fuel blending agents or in the flavor and fragrance industry. Recent efforts at strain optimization have focused on cofactor balance, as fatty acid-derived pathways face the systematic metabolic challenge of net NADPH consumption [in large part, resulting from the key fatty acid biosynthetic enzyme FabG (β-ketoacyl-ACP reductase)] and net NADH production. In this study, we attempted to mitigate cofactor imbalance by heterologously expressing NADH-dependent, rather than NADPH-dependent, versions of FabG identified in previous studies. Of the four NADH-dependent versions of FabG tested in our previously best-reported methyl ketone-producing strain (EGS1895), the version from Acholeplasma laidlawii (Al_FabG) showed the greatest increase in methyl ketone yield in shake flasks (35-75% higher than for an RFP negative-control strain, depending on sugar loading). An improved strain (EGS2920) attained methyl ketone titers during fed-batch fermentation of 5.4 ± 0.5 g/L, which were, on average, ca. 40% greater than those for the base strain (EGS1895) under fermentation conditions optimized in this study. Shotgun proteomic data for strains EGS2920 and EGS1895 during fed-batch fermentation were consistent with the goal of alleviating NADPH limitation through expression of Al_FabG. For example, relative to strain EGS1895, strain EGS2920 significantly upregulated glucose-6-phosphate isomerase (directing flux into glycolysis rather than the NADPH-producing pentose phosphate pathway) and downregulated MaeB (a NADP+-dependent malate dehydrogenase). Overall, the results suggest that heterologous expression of NADH-dependent FabG in E. coli may improve sustained production of fatty acid-derived renewable fuels and chemicals. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-07T06:55:22.20236-05:0
      DOI: 10.1002/bit.26558
  • Split trehalase as a versatile reporter for a wide range of biological
    • Authors: Marija Drikic; Jeroen De Buck
      Abstract: In health care, biosensors are envisioned as universal diagnostic devices with AAAA characteristics (i.e. available for anything, anywhere, anytime, to anyone). Despite numerous attempts to develop such a diagnostic device, none have managed to fulfill all 4 criteria and be commercialized. Glucometers, the most successful class of biosensor currently marketed monitor blood glucose concentrations. Their performance in clinical samples, including sensitivity and specificity, has been optimized and they are small and relatively inexpensive. We aimed to develop a technology that uses this existing biosensor, but adds versatility in detection of a wide range of analytes. Herein, we report the periplasmic trehalase of E. coli as a novel split enzyme reporter capable of converting a wide variety of analytes into glucose. Conditional complementation of trehalase fragments induced by detection of analytes, resulting in trehalose hydrolysis and glucose production, was used to detect antibodies and bacterial cells. We also demonstrated retention of split TreA activity in undiluted clinical samples. In conclusion, a trehalase-based biosensor platform offers a versatile and convenient method for point-of-care applications as it does not require sample preparation or handling and can be integrated with existing glucometers or sensors. This article is protected by copyright. All rights reserved
      PubDate: 2018-02-07T06:50:21.858316-05:
      DOI: 10.1002/bit.26556
  • Multi-facet Implications of PEGylated Lysozyme Stabilized-Silver
           Nanoclusters Loaded Recombinant PTEN Cargo in Cancer Theranostics
    • Authors: Neha Arora; S Lalitha Gavya, Siddhartha Sankar Ghosh
      Abstract: Amalgamation of delivery and tracking of therapeutically relevant moieties on a single platform is made possible by the application of metal nanoclusters, an innovative class of luminescent nanomaterials. Metal nanoclusters, possessing molecule-like attributes, display extraordinary size and shape tunable properties befitting theranostic applications. Herein, we report successful assembly of therapeutically significant phosphatase protein PTEN and fluorescent lysozyme-stabilized silver nanoclusters to accomplish delivery and tracking of the protein. Down-regulation of PTEN perturbs the cellular networking leading to copious pathological conditions. The integration of purified recombinant PTEN with silver nanoclusters was evaluated by fluorescence spectroscopy study. A key feature of this study is the use of polyethylene glycol coating that allows fabrication of the assembly into spherical nanocomposites as characterized by transmission electron microscope along with retention of both optical functionality of the cluster and biological activity of the protein. Prior to cellular application, the functional integrity of PTEN in the composite was determined in vitro, by enzymatic assay employing para-nitrophenylphosphate as substrate. Cellular internalization of the cargo was studied by confocal microscopy and flow cytometry analysis. The efficacy of the payload on modulation of cellular signaling was assessed on cell lines that expressed PTEN differentially. PTEN null U-87 MG and PTEN expressing MCF7 cell lines displayed successful alteration of AKT and FAK signaling proteins culminating in cell cycle arrest and reduced wound healing capacity. A dose dependent reduction in cell proliferation of MCF7 cells was achieved. For U-87 MG, treatment with the payload resulted in chemosensitization towards anti-cancer drug erlotinib. Thus, PEG coated GST-PTEN loaded silver nanoclusters serves as a comprehensive system encompassing cellular imaging and protein delivery with potential biomedical implications. This article is protected by copyright. All rights reserved
      PubDate: 2018-01-31T06:35:47.339968-05:
      DOI: 10.1002/bit.26553
  • Improving the Recombinant Human Erythropoietin Glycosylation through
           Microsomes in CHO Cell-Free System
    • Authors: Chandrasekhar Gurramkonda; Aniruddha Rao, Shayan Borhani, Manohar Pilli, Sevda Deldari, Xudong Ge, Niloufar Pezeshk, Tzu-Chiang Han, Michael Tolosa, Yordan Kostov, Leah Tolosa, David W. Wood, Krishna Vattem, Douglas D. Frey, Govind Rao
      Abstract: Cell-Free Protein Synthesis (CFPS) offers many advantages for the production of recombinant therapeutic proteins using the CHO cell-free system. However, many complex proteins are still difficult to express using this method. To investigate the current bottlenecks in cell-free glycoprotein production, we chose erythropoietin (40% glycosylated), an essential endogenous hormone which stimulates the development of red blood cells. Here, we report the production of recombinant erythropoietin (EPO) using CHO cell-free system. Using this method, EPO was expressed and purified with a 2-fold increase in yield when the cell-free reaction was supplemented with CHO microsomes. The protein was purified to near homogeneity using an ion-metal affinity column. We were able to analyze the expressed and purified products (glycosylated cell-free EPO runs at 25-28 kDa, and unglycosylated protein runs at 20 kDa on an SDS-PAGE), identifying the presence of glycan moieties by PNGase shift assay. The purified protein was predicted to have ∼2300 IU in vitro activity. Additionally, we tested the presence and absence of sugars on the cell-free EPO using a lectin-based assay system. The results obtained in this study indicate that microsomes augmented in vitro production of the glycoprotein is useful for the rapid production of single doses of a therapeutic glycoprotein drug and to rapidly screen glycoprotein constructs in the development of these types of drugs. CFPS is useful for implementing a lectin-based method for rapid screening and detection of glycan moieties, which is a critical quality attribute in the industrial production of therapeutic glycoproteins. This article is protected by copyright. All rights reserved
      PubDate: 2018-01-31T06:35:34.245964-05:
      DOI: 10.1002/bit.26554
  • Application of hydrocarbon and perfluorocarbon oxygen vectors to enhance
           heterologous production of hyaluronic acid in engineered Bacillus subtilis
    • Authors: Adam W. Westbrook; Xiang Ren, Murray Moo-Young, C. Perry Chou
      Abstract: In microbial cultivations for hyaluronic acid (HA) production, oxygen can be a limiting substrate due to its poor solubility in aqueous medium and the substantial increase in culture viscosity at relatively low HA titers. Shear stress due to the high agitation and aeration rates required to overcome oxygen limitation may reduce the quality (i.e. molecular weight) of HA, and production costs associated with power consumption and supplemental oxygen may be excessive. Here, we report the application of oxygen vectors to the heterologous production of HA in engineered Bacillus subtilis, leading to significantly improved culture performance. We first derived an improved HA-producing strain of B. subtilis through engineering of the promoter driving coexpression of seHas and tuaD, leading to high-level HA production. Out of seven potential oxygen vectors evaluated in a preliminary screening, significant improvements to the HA titer and/or cell density were observed in cultures containing n-heptane, n-hexadecane, perfluoromethyldecalin, and perfluoro-1,3-dimethylcyclohexane. Adjustments to the vector concentration, timing of vector addition, and the agitation rate resulted in further enhancements, with the HA titer reaching up to 4.5 g/L after only 10 h cultivation. Moreover, our results indicate that certain vectors may alter the functional expression of Class I hyaluronan synthase (HAS) in B. subtilis, and that higher shear rates may drive more carbon flux through the HA biosynthetic pathway without negatively affecting the MW. Our study demonstrates the efficacy of oxygen vectors to enhance heterologous HA production in B. subtilis, and provides valuable insight for future bioprocess development in microbial HA production. This article is protected by copyright. All rights reserved
      PubDate: 2018-01-31T05:30:24.637406-05:
      DOI: 10.1002/bit.26551
  • Cover Image, Volume 115, Number 3, March 2018
    • Authors: Pia Gruber; Filipe Carvalho, Marco P. C. Marques, Brian O'Sullivan, Fabiana Subrizi, Dragana Dobrijevic, John Ward, Helen C. Hailes, Pedro Fernandes, Roland Wohlgemuth, Frank Baganz, Nicolas Szita
      Abstract: The cover image, by Pia Gruber et al., is based on the Article Enzymatic synthesis of chiral amino-alcohols by coupling transketolase and transaminase-catalyzed reactions in a cascading continuous-flow microreactor system,
      DOI : 10.1002/bit.26470. Design Credit: Winter Mraz, Frozen Ink Designs.
      PubDate: 2018-01-29T18:12:27.973341-05:
  • Glutamine synthetase gene knockout-human embryonic kidney 293E cells for
           stable production of monoclonal antibodies
    • Authors: Da Young Yu; Sang Yoon Lee, Gyun Min Lee
      Abstract: Previously, it was inferred that a high glutamine synthetase (GS) activity in human embryonic kidney (HEK) 293E cells results in elevated resistance to methionine sulfoximine (MSX) and consequently hampers GS-mediated gene amplification and selection by MSX. To overcome this MSX resistance in HEK293E cells, a GS-knockout HEK293E cell line was generated using the CRISPR/Cas9 system to target the endogenous human GS gene. The GS-knockout in the HEK293E cell line (RK8) was confirmed by Western blot analysis of GS and by observation of glutamine-dependent growth. Unlike the wild type HEK293E cells, the RK8 cells were successfully used as host cells to generate a recombinant HEK293E cell line (rHEK293E) producing a monoclonal antibody (mAb). When the RK8 cells were transfected with the GS expression vector containing the mAb gene, rHEK293E cells producing the mAb could be selected in the absence as well as in the presence of MSX. The gene copies and mRNA expression levels of the mAb in rHEK293E cells were also quantified using qRT-PCR. Taken together, the GS-knockout HEK293E cell line can be used as host cells to generate stable rHEK293E cells producing a mAb through GS-mediated gene selection in the absence as well as in the presence of MSX. This article is protected by copyright. All rights reserved
      PubDate: 2018-01-23T05:45:46.5229-05:00
      DOI: 10.1002/bit.26552
  • Correlation of simulation/finite element analysis to the separation of
           intrinsically magnetic spores and red blood cells using a microfluidic
           magnetic deposition system
    • Authors: Jianxin Sun; L. Moore, Wei Xue, James Kim, Maciej Zborowski, Jeffrey J Chalmers
      Abstract: Magnetic separation of cells has been, and continues to be, widely used in a variety of applications, ranging from healthcare diagnostics to detection of food contamination. Typical, these technologies require cells labeled with antibody magnetic particle conjugate and a high magnetic energy gradient created in the flow containing the labeled cells (i.e. a column packed with magnetically inducible material), or dense packing of magnetic particles next to the flow cell. Such designs while creating high magnetic energy gradients, are not amenable to easy, highly detailed, mathematic characterization.Our laboratories have been characterizing and developing analysis and separation technology that can be used on intrinsically magnetic cells or spores which are typically orders of magnitude weaker than typically immunomagnetically labeled cells. One such separation system is magnetic deposition microscopy (MDM) which not only separates cells, but which deposits cells in specific locations on slides for further microscopic analysis. In this study, the MDM system has been further characterized, using finite element and computational fluid mechanics software, and separation performance predicted, using a model which combines: 1) the distribution of the intrinsic magnetophoretic mobility of the cells (spores), 2) the fluid flow within the separation device, 3) accurate maps of the values of the magnetic field (max 2.27 T), and magnetic energy gradient (max of 4.41 T2/mm) within the system. Guided by this model, experimental studies indicated that greater than 95 percent of the intrinsically magnetic bacillus spores can be separated with the MDM system. Further, this model allows analysis of cell trajectories which can assist in the design of higher throughput systems. This article is protected by copyright. All rights reserved
      PubDate: 2018-01-16T07:25:51.023585-05:
      DOI: 10.1002/bit.26550
  • Inhibition of Apoptosis Using Exosomes in Chinese Hamster Ovary Cell
    • Authors: Seora Han; Won Jong Rhee
      Abstract: Animal cell culture technology for therapeutic protein production has shown significant improvement over the last few decades. Chinese hamster ovary (CHO) cells have been widely adapted for the production of biopharmaceutical drugs. In the biopharmaceutical industry, it is crucial to develop cell culture media and culturing conditions to achieve the highest productivity and quality. However, CHO cells are significantly affected by apoptosis in the bioreactors, resulting in a substantial decrease in product quantity and quality. Thus, to overcome the obstacle of apoptosis in CHO cell culture, it is critical to develop a novel method that does not have minimal concern of safety or cost. Herein, we showed for the first time that exosomes, which are nano-sized extracellular vesicles, derived from CHO cells inhibited apoptosis in CHO cell culture when supplemented to the culture medium. Flow cytometric and microscopic analyses revealed that substantial amounts of exosomes were delivered to CHO cells. Higher cell viability after staurosporine treatment was observed by exosome supplementation (67.3%) as compared to control (41.1%). Furthermore, exosomes prevented the mitochondrial membrane potential loss and caspase-3 activation, meaning that the exosomes enhanced cellular activities under pro-apoptotic condition. As the exosomes supplements are derived from CHO cells themselves, it is not only beneficial for the biopharmaceutical productivity of CHO cell culture to inhibit apoptosis, but also from a regulatory standpoint to diminish any safety concerns. Thus, we conclude that the method developed in this research may contribute to the biopharmaceutical industry where minimizing apoptosis in CHO cell culture is beneficial. This article is protected by copyright. All rights reserved
      PubDate: 2018-01-16T07:21:10.241642-05:
      DOI: 10.1002/bit.26549
  • Effective Role of Medium Supplementation in Microalgal Lipid Accumulation
    • Authors: Azadeh Fazeli Danesh; Peter Mooij, Sirous Ebrahimi, Robbert Kleerebezem, Mark Loosdrecht
      Abstract: The present study investigated the interaction between starch and lipid accumulation was in a green microalgae enrichment culture. The objective was to optimize the lipid content by manipulation of the medium in regular batch culture. Two medium designs were evaluated: first a high ortho-P concentration with vitamin supplement (Pi-vitamins supplemented medium), second normal growth medium (control). Both media contained a low amount of nitrogen which was consumed during batch growth in three days. The batch experiments were prolonged continued for another four days with the absence of soluble nitrogen in the medium. When the mixed microalgal culture was incubated with in the Pi-vitamin supplemented medium, the lipid and starch content of the culture increased within the first three days to 102.0  ± 5.2 mg.l−1 (12.7 ± 0.6% of DW) and 31.7 ± 1.6 mg.l−1 (4.0 ± 0.2% of DW), respectively. On the last day of the experiment, the lipid and starch content in Pi-vitamin medium increased to 663.1 ± 32.5 (33.4 ± 1.6% of DW) and 127.5 ± 5.2 (6.4 ± 0.3% of DW) mg.l−1. and However, the lipid and starch content in the control process, reached to 334.7 ± 16.4 (20.1 ± 1.0% of DW) and 94.3 ± 4.6 (5.7 ± 0.3% of DW), respectively. The high Pi-vitamin medium induced storing lipid formation clearly while the starch formation was not affected. The lipid contents reported here are among the high reported in the literature, note that already under full growth conditions significant lipid levels occurred in the algal enrichment culture. The high lipid productivity of the reported mixed microalgae culture provides an efficient route for efficient algal biodiesel production. This article is protected by copyright. All rights reserved
      PubDate: 2018-01-16T07:20:23.473893-05:
      DOI: 10.1002/bit.26548
  • Ultra scale-down approaches to study the centrifugal harvest for viral
           vaccine production
    • Authors: Beatrice J Melinek; Sandrine Dessoy, Bernice Wright, Dan G Bracewell, Tarit K Mukhopadhyay
      Abstract: Large scale continuous cell-line cultures promise greater reproducibility and efficacy for the production of influenza vaccines, and adenovirus for gene therapy. This paper seeks to use an existing validated ultra scale-down tool, which is designed to mimic the commercial scale process environment using only millilitres of material, to provide some initial insight into the performance of the harvest step for these processes. The performance of industrial scale centrifugation and subsequent downstream process units is significantly affected by shear. The properties of these cells, in particular their shear sensitivity, may be changed considerably by production of a viral product, but literature on this is limited to date. In addition, the scale-down tool used here has not previously been applied to the clarification of virus production processes. The results indicate that virus infected cells do not actually show any increase in sensitivity to shear, and may indeed become less shear sensitive, in a similar manner to that previously observed in old or dead cell cultures. Clarification may be most significantly dependent on the virus release mechanism, with the budding influenza virus producing a much greater decrease in clarification than the lytic, non-enveloped adenovirus. A good match was also demonstrated to the industrial scale performance in terms of clarification, protein release and impurity profile. This article is protected by copyright. All rights reserved
      PubDate: 2018-01-08T07:25:23.095928-05:
      DOI: 10.1002/bit.26546
  • Enrichment of high affinity subclasses and glycoforms from serum-derived
           IgG using FcγRs as affinity ligands
    • Authors: Boesch AW; Kappel JH, Mahan AE, Chu TH, Crowley AR, Osei-Owusu NY, Alter G, Ackerman ME
      Abstract: As antibodies continue to gain predominance in drug discovery and development pipelines, efforts to control and optimize their activity in vivo have matured to incorporate sophisticated abilities to manipulate engagement of specific Fc binding partners. Such efforts to promote diverse functional outcomes include modulating IgG-Fc affinity for FcγRs to alternatively potentiate or reduce effector functions, such as antibody-dependent cellular cytotoxicity and phagocytosis. While a number of natural and engineered Fc features capable of eliciting variable effector functions have been demonstrated in vitro and in vivo, elucidation of these important functional relationships has taken significant effort through use of diverse genetic, cellular and enzymatic techniques. As an orthogonal approach, we demonstrate use of FcγR as chromatographic affinity ligands to enrich and therefore simultaneously identify favored binding species from a complex mixture of serum-derived pooled polycloncal human IgG, a load material that contains the natural repertoire of Fc variants and post-translational modifications. The FcγR-enriched IgG was characterized for subclass and glycoform composition and the impact of this bioseparation step on antibody activity was measured in cell-based effector function assays including Natural Killer cell activation and monocyte phagocytosis. This work demonstrates a tractable means to rapidly distinguish complex functional relationships between two or more interacting biological agents by leveraging affinity chromatography followed by secondary analysis with high-resolution biophysical and functional assays and emphasizes a platform capable of surveying diverse natural post-translational modifications that may not be easily produced with high purity or easily accessible with recombinant expression techniques. This article is protected by copyright. All rights reserved
      PubDate: 2018-01-08T07:20:29.236511-05:
      DOI: 10.1002/bit.26545
  • Isotype dependent on-column non-reversible aggregation of monoclonal
    • Authors: Monika Farys; Daniel Gibson, Alan Lewis, Will Lewis, Richard Kucia-Tran
      Abstract: Monoclonal antibodies of the IgG2 and IgG4 isotype were found to exhibit an increased propensity for displaying two-peak elution profiles during cation exchange chromatography. In some cases, this two-peak elution profile also resulted in the formation of non-reversible mAb aggregates.Comparison of IgG1, IgG2 and IgG4 molecules with the same variable region reveals that the two-peak behaviour is predominantly mediated by the constant region and most likely the lower CH1, hinge and upper CH2 regions of the mAb. Furthermore, comparison of the behaviour of two different IgG4 molecules, reveals that the degree of non-reversible aggregate formation, whilst facilitated by the isotype format, is mediated primarily by the variable region of the molecule. As well as the properties of the mAb molecule itself, the chemistry and structure of the cation exchange resin was also found to have an effect, with the two-peak elution profile being more pronounced with polymer-grafted resins such as Capto S Impact and Eshmuno CPX.These results combined support the theory that binding of IgG2 and IgG4 mAbs to cation exchange resins usually occurs through at least two mechanisms mediated by the structural features of the constant region of IgG2s and IgG4s. One of these mechanisms is not only stronger than the other, but also can lead to a conformational change in the molecule. This conformational change can occur in both variable and constant domain of the antibody. This transitory unfolded state can in turn lead to non-reversible aggregation of some mAb molecules. This article is protected by copyright. All rights reserved
      PubDate: 2018-01-08T07:20:27.248371-05:
      DOI: 10.1002/bit.26547
  • Development of a temperature-responsive yeast cell factory using
           engineered Gal4 as a protein switch
    • Authors: Pingping Zhou; Wenping Xie, Zhen Yao, Yongqiang Zhu, Lidan Ye, Hongwei Yu
      Abstract: Conflict between cell growth and product accumulation is frequently encountered in biosynthesis of secondary metabolites. Herein, a temperature-dependent dynamic control strategy was developed by modifying the GAL regulation system to facilitate two-stage fermentation in yeast. A temperature-sensitive Gal4 mutant Gal4M9 was created by directed evolution, and used as a protein switch in ΔGAL80 yeast. After EGFP-reported validation of its temperature-responsive induction capability, the sensitivity and stringency of this system in multi-gene pathway regulation was tested, using lycopene as an example product. When Gal4M9 was used to control the expression of PGAL-driven pathway genes, growth and production was successfully decoupled upon temperature shift during fermentation, accumulating 44% higher biomass and 177% more lycopene than the control strain with wild-type Gal4. This is the first example of adopting temperature as an input signal for metabolic pathway regulation in yeast cell factories. This article is protected by copyright. All rights reserved
      PubDate: 2018-01-08T07:05:24.761723-05:
      DOI: 10.1002/bit.26544
  • Using extensional flow to reveal diverse aggregation landscapes for three
           IgG1 molecules
    • Authors: Leon F. Willis; Amit Kumar, John Dobson, Nick Bond, David Lowe, Richard Turner, Sheena E. Radford, Nikil Kapur, David J. Brockwell
      Abstract: Monoclonal antibodies (mAbs) currently dominate the biopharmaceutical sector due to their potency and efficacy against a range of disease targets. These proteinaceous therapeutics are, however, susceptible to unfolding, mis-folding and aggregation by environmental perturbations. Aggregation thus poses an enormous challenge to biopharmaceutical development, production, formulation and storage. Hydrodynamic forces have also been linked to aggregation, but the ability of different flow fields (e.g. shear and extensional flow) to trigger aggregation has remained unclear. To address this question, we previously developed a device that allows the degree of extensional flow to be controlled. Using this device we demonstrated that mAbs are particularly sensitive to the force exerted as a result of this flow-field. Here, to investigate the utility of this device to bio-process / biopharmaceutical development, we quantify the effects of the flow field and protein concentration on the aggregation of three mAbs. We show that the response surface of mAbs is distinct from that of bovine serum albumin (BSA) and also that mAbs of similar sequence display diverse sensitivity to hydrodynamic flow. Finally, we show that flow-induced aggregation of each mAb is ameliorated by different buffers, opening up the possibility of using the device as a formulation tool. Perturbation of the native state by extensional flow may thus allow identification of aggregation-resistant mAb candidates and their bio-process parameters and formulation to be optimized earlier in the drug-discovery pipeline using sub-milligram quantities of material. This article is protected by copyright. All rights reserved
      PubDate: 2018-01-08T06:40:47.625744-05:
      DOI: 10.1002/bit.26543
  • Role of Respiratory Terminal Oxidases in the Extracellular Electron
           Transfer Ability of Cyanobacteria
    • Authors: Narendran Sekar; Jian Wang, Yan Zhou, Yi Fang, Yajun Yan, Ramaraja P. Ramasamy
      Abstract: Cyanobacteria are used as anode catalysts in photo-bioelectrochemical cells to generate electricity in a sustainable, economic and environmental friendly manner using only water and sunlight. Though cyanobacteria (CB) possess unique advantage for solar energy conversion by virtue of its robust photosynthesis, they cannot efficiently perform extracellular electron transfer (EET). The reasons being, unlike dissimilatory metal reducing bacteria (that are usually exploited in microbial fuel cells to generate electricity), (1) CB do not possess any special features on their outer membrane to carry out EET and (2) the electrons generated in photosynthetic electron transport chain are channeled into competing respiratory pathways rather than to the anode. While, CB, genetically engineered to express outer membrane cytochrome S (OmcS), was found to generate ∼ nine-fold higher photocurrent compared to that of wild-type cyanobacterium in our previous work, the role of respiratory terminal oxidases (RTOs) of cyanobacteria for EET had not been much explored. In this study, each of the three RTOs in Synechococcus elongatus PCC7942 namely such as bd-type quinol oxidase, aa3-type cytochrome oxidase and cbb3-type cytochrome oxidase was knocked-out one at a time (cyd-, cox- and cco- respectively) and its contribution for extracellular ferricyanide reduction and photocurrent generation were investigated. The knock-out mutant lacking functional bd-type quinol oxidase (cyd-) exhibited greater EET by reducing more ferricyanide compared to other single knock-out mutants as well as the wild type. Further, cyd-omcs (the cyd- mutant expressing OmcS) was found to generate more photocurrent than the corresponding single knock out controls and the wild type. This study clearly demonstrates that the bd-quinol oxidase diverted more electrons from the photosynthetic electron transport chain towards respiratory oxygen reduction and knocking it out had certainly enhanced the cyanobacterial EET. This article is protected by copyright. All rights reserved
      PubDate: 2018-01-08T06:40:45.351652-05:
      DOI: 10.1002/bit.26542
  • Consolidated bioprocessing of lignocellulosic biomass to lactic acid by a
           synthetic fungal-bacterial consortium
    • Authors: Robert Lawrence Shahab; Jeremy S. Luterbacher, Simone Brethauer, Michael Hans-Peter Studer
      Abstract: Consolidated bioprocessing (CBP) of lignocellulosic feedstocks to platform chemicals requires complex metabolic processes, which are commonly executed by single genetically engineered microorganisms. Alternatively, synthetic consortia can be employed to compartmentalize the required metabolic functions among different specialized microorganisms as demonstrated in this work for the direct production of lactic acid from lignocellulosic biomass.We composed an artificial cross-kingdom consortium and co-cultivated the aerobic fungus Trichoderma reesei for the secretion of cellulolytic enzymes with facultative anaerobic lactic acid bacteria. We engineered ecological niches to enable the formation of a spatially structured biofilm. Up to 34.7 gL−1 lactic acid could be produced from 5% (w/w) microcrystalline cellulose.Challenges in converting pretreated lignocellulosic biomass include the presence of inhibitors, the formation of acetic acid and carbon catabolite repression. In the CBP consortium hexoses and pentoses were simultaneously consumed and metabolic cross-feeding enabled the in situ degradation of acetic acid. As a result, superior product purities were achieved and 19.8 gL−1 (85.2% of the theoretical maximum) of lactic acid could be produced from non-detoxified steam-pretreated beech wood. These results demonstrate the potential of consortium-based CBP technologies for the production of high value chemicals from pretreated lignocellulosic biomass in a single step. This article is protected by copyright. All rights reserved
      PubDate: 2018-01-08T06:40:40.794471-05:
      DOI: 10.1002/bit.26541
  • Selective production of decanoic acid from iterative reversal of
           β-oxidation pathway
    • Authors: Seohyoung Kim; Ramon Gonzalez
      Abstract: Decanoic acid is a valuable compound used as precursor for industrial chemicals, pharmaceuticals and biofuels. Despite efforts to produce it from renewables, only limited achievements have been reported. Here, we report an engineered cell factory able to produce decanoic acid as a major product from glycerol, and abundant and renewable feedstock. We exploit the overlapping chain-length specificity of β-oxidation reversal (r-BOX) and thioesterase enzymes to selectively generate decanoic acid. This was achieved by selecting r-BOX enzymes that support the synthesis of acyl-CoA of up to 10 carbons (thiolase BktB and enoyl-CoA reductase EgTER) and a thioesterase that exhibited high activity towards decanoyl-CoA and longer-chain acyl-CoAs (FadM). Combined chromosomal and episomal expression of r-BOX core enzymes such as enoyl-CoA reductase and thiolase (in the presence of E. coli thioesterase FadM) increased titer and yield of decanoic acid, respectively. The carbon flux towards decanoic acid was substantially increased by the use of an organic overlay, which decreased its intracellular accumulation and presumably increased its concentration gradient across cell membrane, suggesting that decanoic acid transport to the extracellular medium might be a major bottleneck. When cultivated in the presence of a n-dodecane overlay, the final engineered strain produced 2.1g/L of decanoic acid with a yield of 0.1g/g glycerol. Collectively, our data suggests that r-BOX can be used as a platform to selectively produce decanoic acid and its derivatives at high yield, titer and productivity. This article is protected by copyright. All rights reserved
      PubDate: 2018-01-08T06:35:55.405024-05:
      DOI: 10.1002/bit.26540
  • Modulation of saturation and chain length of fatty acids in Saccharomyces
           cerevisiae for production of cocoa butter-like lipids
    • Authors: David Bergenholm; Michael Gossing, Yongjun Wei, Verena Siewers, Jens Nielsen
      Abstract: Chain length and degree of saturation plays an important role for the characteristics of various products derived from fatty acids, such as fuels, cosmetics and food additives. The seeds of Theobroma cacao are the source of cocoa butter, a natural lipid of high interest for the food and cosmetics industry. Cocoa butter is rich in saturated fatty acids that are stored in the form of triacylglycerides (TAGs). One of the major TAG species of cocoa butter, consisting of two stearic acid molecules and one oleic acid molecule (stearic acid-oleic acid-stearic acid, sn-SOS), is particularly rare in nature as the saturated fatty acid stearic acid is typically found only in low abundance. Demand for cocoa butter is increasing, yet T. cacao can only be cultivated in some parts of the tropics. Alternative means of production of cocoa butter lipids (CBLs) are therefore sought after. Yeasts also store fatty acids in the form of TAGs, but these are typically not rich in saturated fatty acids. To make yeast an attractive host for microbial production of CBLs, its fatty acid composition needs to be optimized.We engineered Saccharomyces cerevisiae yeast strains towards a modified fatty acid synthesis. Analysis of the fatty acid profile of the modified strains showed that the fatty acid content as well as the titers of saturated fatty acids and the titers of TAGs were increased. The relative content of potential CBLs in the TAG pool reached up to 22% in our engineered strains, which is a 5.8-fold increase over the wild-type. SOS content reached a level of 9.8% in our engineered strains, which is a 48-fold increase over the wild type. This article is protected by copyright. All rights reserved
      PubDate: 2018-01-04T06:20:36.484083-05:
      DOI: 10.1002/bit.26518
  • Biotechnology and Bioengineering: Volume 115, Number 3, March 2018
    • Pages: 519 - 523
      PubDate: 2018-01-29T18:12:24.002052-05:
      DOI: 10.1002/bit.26414
  • Modeling and optimization of lipid accumulation by Yarrowia lipolytica
           from glucose under nitrogen depletion conditions
    • Authors: Carlos Eduardo Robles-Rodríguez; Rafael Muñoz-Tamayo, Carine Bideaux, Nathalie Gorret, Stéphane E. Guillouet, Carole Molina-Jouve, Gilles Roux, César Arturo Aceves-Lara
      Abstract: Oleaginous yeasts have been seen as a feasible alternative to produce the precursors of biodiesel due to their capacity to accumulate lipids as triacylglycerol having profiles with high content of unsaturated fatty acids. The yeast Yarrowia lipolytica is a promising microorganism that can produce lipids under nitrogen depletion conditions and excess of the carbon source. However, under these conditions, this yeast also produces citric acid (overflow metabolism) decreasing lipid productivity. This work presents two mathematical models for lipid production by Yarrowia lipolytica from glucose. The first model is based on Monod and inhibition kinetics, and the second one is based on the Droop quota model approach, which is extended to yeast. The two models showed good agreements with the experimental data used for calibration and validation. The quota based model presented a better description of the dynamics of nitrogen and glucose dynamics leading to a good managing of N/C ratio, which makes this model interesting for control purposes. Then, quota model was used to evaluate, by means of simulation, a scenario for optimizing lipid productivity and lipid content. For that, a control strategy was designed by approximating the flow rates of glucose and nitrogen with piecewise linear functions. Simulation results achieved a productivity of 0.95 g.L−1.h−1 and a lipid content fraction of 0.23 g.g−1, which indicate a promising alternative for the optimization of lipids production. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-30T07:25:48.185496-05:
      DOI: 10.1002/bit.26537
  • A robust protocol for directed aryl sulfotransferase evolution towards the
           carbohydrate building block GlcNAc
    • Authors: Shohana S. Islam; Diana M. Mate, Ronny Martínez, Felix Jakob, Ulrich Schwaneberg
      Abstract: Bacterial aryl sulfotransferases (AST) utilize p-nitrophenylsulfate (pNPS) as a phenolic donor to sulfurylate typically a phenolic acceptor. Interest in aryl sulfotransferases is growing because of their broad variety of acceptors and cost-effective sulfuryl-donors. For instance, aryl sulfotransferase A (ASTA) from Desulfitobacterium hafniense was recently reported to sulfurylate D-glucose. In this study, a directed evolution protocol was developed and validated for aryl sulfotransferase B (ASTB). Thereby the well-known pNPS quantification system was advanced to operate efficiently as a continuous screening system in 96-well MTP format with a true coefficient of variation of 14.3%. A random mutagenesis library (SeSaM library) of ASTB was screened (1,760 clones) to improve sulfurylation of the carbohydrate building block N-acetylglucosamine (GlcNAc). The beneficial variant ASTB-V1 (Val579Asp) showed an up to 3.4-fold increased specific activity towards GlcNAc when compared to ASTB-WT. HPLC- and MS-analysis confirmed ASTB-V1's increased GlcNAc monosulfurylation (2.4-fold increased product formation) representing the validation of the first successful directed evolution round of an AST for a saccharide substrate. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-30T07:25:42.723346-05:
      DOI: 10.1002/bit.26535
  • From lab to market: An integrated bioprocess design approach for
           new-to-nature biosurfactants produced by Starmerella bombicola
    • Authors: Lisa Van Renterghem; Sophie L.K. W. Roelants, Niki Baccile, Katrijn Uyttersprot, Marie Claire Taelman, Bernd Everaert, Stein Mincke, Sam Ledegen, Sam Debrouwer, Kristel Scholtens, Christian Stevens, Wim Soetaert
      Abstract: Glycolipid microbial biosurfactants, like sophorolipids (SLs), generate high industrial interest as 100% biobased alternatives for traditional surfactants. A well-known success story is the efficient SL producer Starmerella bombicola, which reaches titers well above 200 g/L. Recent engineering attempts have enabled the production of completely new types of molecules by S. bombicola, like the ‘non-symmetrical bolaform (nsBola) SLs’. As classic SLs are mostly applied in eco-friendly detergents, the possible use of these bolaform SLs in detergent applications was evaluated by scaling up the production process (150 L) and evaluating the purified product. This paper shows that they can be used as green and non-irritant surfactants in for example (automatic) dishwasher applications. However, the limited chemical stability at higher pH values (> 6.5), due to the presence of an ester function in the biosurfactant molecule, is a major drawback that will most likely inhibit market introduction. An integrated bioprocess design was thus applied to resolve this issue. The strategy was to replace the fed fatty acids, responsible for the ester bond in nsBola SLs, with fatty alcohols, to generate so-called ‘symmetrical bolaform (sBola) SLs’, containing two instead of one glycosidic bond. This requires a change in the feeding strategy, but also the blocking the fatty alcohols from metabolizing/oxidizing through the suggested ω-oxidation pathway. Two putative fatty alcohol oxidase genes (fao1 and fao2) were identified in the S. bombicola genome and deleted in the nsBola SL producing strain (ΔatΔsble). Shake flask experiments for these new strains (ΔatΔsbleΔfoa1 and ΔatΔsbleΔfoa2) were performed to evaluate if the fed fatty alcohols were directly implemented into the SL biosynthesis pathway. Indeed, sBola SL production up to 20 g/L was observed for the ΔatΔsbleΔfao1 strain, while the ΔatΔsbleΔfao2 strain only produced nsBola SLs. The sBola SLs were purified and their symmetrical structure was confirmed by NMR. They were found to be significantly more stable at higher pH, opening up the application potential of the biosurfactant by enhancing its stability properties. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-30T07:25:29.44709-05:0
      DOI: 10.1002/bit.26539
  • High titer oncolytic measles production process by integration of
           Dielectric spectroscopy as online monitoring system
    • Authors: Tanja A. Grein; Daniel Loewe, Hauke Dieken, Denise Salzig, Tobias Weidner, Peter Czermak
      Abstract: Oncolytic viruses offer new hope to millions of patients with incurable cancer. One promising class of oncolytic viruses is Measles virus, but its broad administration to cancer patients is currently hampered by the inability to produce the large amounts of virus needed for treatment (1010-1012 virus particles per dose). Measles virus is unstable, leading to very low virus titers during production. The time of infection and time of harvest are therefore critical parameters in a Measles virus production process, and their optimization requires an accurate online monitoring system. We integrated a probe based on dielectric spectroscopy (DS) into a stirred tank reactor to characterize the Measles virus production process in adherent growing Vero cells. We found that DS could be used to monitor cell adhesion on the microcarrier and that the optimal virus harvest time correlated with the global maximum permittivity signal. In 16 independent bioreactor runs, the maximum Measles virus titer was achieved approximately 40 h after the permittivity maximum. Compared to an uncontrolled Measles virus production process, the integration of DS increased the maximum virus concentration by more than three orders of magnitude. This was sufficient to achieve an active Measles virus concentration of >1010 TCID50 ml−1. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-30T07:25:24.37612-05:0
      DOI: 10.1002/bit.26538
  • Gold nanorod-photosensitizer conjugates with glutathione-sensitive
           linkages for synergistic cancer photodynamic/photothermal therapy
    • Authors: Jongseon Choi; Sangeun Lee, Jeong-Sook Park, So Yeon Kim
      Abstract: Recently, photodynamic therapy (PDT) has been intensively investigated as a useful modality for the treatment of various cancers. In addition, near infrared (NIR) photothermal therapy (PTT) using gold nanocarriers has attracted particular interest as a hyperthermia strategy. In this study, gold nanorod (AuNR)-photosensitizer conjugates with glutathione-sensitive linkages were designed for PDT and PTT. Several kinds of AuNRs with different aspect ratios were synthesized and modified with FA-conjugated block copolymers (FA-PEG-P(Asp)-DHLA) and Chlorine6 (Ce6) as a photosensitizer. The surface-modified AuNRs showed excellent stability and solubility in aqueous solution. In particular, FA-PEG-P(Asp)-DHLA-AuNR100-SS-Ce6 with a 3.84 aspect ratio exhibited strong photothermal effects, enhanced singlet oxygen generation, and marked phototoxicity. Based on these results, we suggest that AuNR-photosensitizer conjugates with glutathione-sensitive linkages have potential application in PDT/PTT for effective clinical treatment of various cancers. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-30T07:20:22.672684-05:
      DOI: 10.1002/bit.26536
  • Protein-Engineering of Chitosanase from Bacillus sp. MN to Alter its
           Substrate Specificity
    • Authors: Eva K. Regel; Tobias Weikert, Anna Niehues, Bruno M. Moerschbacher, Ratna Singh
      Abstract: Partially acetylated chitosan oligosaccharides (paCOS) have various potential applications in agriculture, biomedicine and pharmaceutics due to their suitable bioactivities. One method to produce paCOS is partial chemical hydrolysis of chitosan polymers, but that leads to poorly defined mixtures of oligosaccharides. However, the effective production of defined paCOS is crucial for fundamental research and for developing applications. A more promising approach is enzymatic depolymerization of chitosan using chitinases or chitosanases, as the substrate specificity of the enzyme determines the composition of the oligomeric products. Protein-engineering of these enzymes to alter their substrate specificity can overcome the limitations associated with naturally occurring enzymes and expand the spectrum of specific paCOS that can be produced. Here, engineering the substrate specificity of Bacillus sp. MN chitosanase is described for the first time. Two muteins with active site substitutions can accept N-acetyl-D-glucosamine units at their subsite (−2), which is impossible for the wildtype enzyme. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-27T07:27:19.233032-05:
      DOI: 10.1002/bit.26533
  • Identification of process conditions influencing protein aggregation in
           Chinese hamster ovary cell culture
    • Authors: Albert Jesuran Paul; René Handrick, Sybille Ebert, Friedemann Hesse
      Abstract: Protein aggregation of monoclonal antibodies (mAbs) is a common phenomenon associated with the production of these biopharmaceuticals. These aggregates can lead to adverse side effects in patients upon administration, thus expensive downstream processing steps to remove the higher molecular weight species are inevitable. A preferable approach is to reduce the level of aggregation during bioprocessing by a careful adjustment of critical process parameters. Recently, new analytical methods enabled characterization of mAb aggregation during bioprocessing of mammalian cells. Furthermore, rapid and efficient bioprocess optimization has been performed using design of experiments (DoE) strategies. In this work, we describe a DoE-based approach for the analysis of process parameters and cell culture additives influencing protein aggregation in Chinese hamster ovary (CHO) cell cultures. Important bioprocess variables influencing the aggregation of mAb and host cell proteins were identified in initial screening experiments. Response surface modeling was further applied in order to find optimal conditions for the reduction of protein aggregation during cell culture. It turned out that a temperature-shift to 31 °C, osmolality above 420 mOsm/kg, agitation at 100 rpm and 0.04% (w/v) antifoam significantly reduced the level of aggregates without substantial detrimental effects on cell culture performance in our model system. Finally, the aggregation reducing conditions were verified and applied to another production system using a different bioprocess medium and another CHO cell line producing another mAb. Our results show that protein aggregation can be controlled during cell culture and helps to improve bioprocessing of mAbs, by giving insights into the protein aggregation at its origin in mammalian cell culture. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-27T07:22:11.882306-05:
      DOI: 10.1002/bit.26534
  • Precision-Porous Templated Scaffolds of Varying Pore Size Drive Dendritic
           Cell Activation
    • Authors: Ruying Chen; Hongyan Ma, Lei Zhang, James D. Bryers
      Abstract: Scaffold based systems have shown significant potential in modulating immune responses in vivo. While there has been much attention on macrophage interactions with tissue engineered scaffolds for tissue regeneration, fewer studies have looked at the effects of scaffold design on the response of immune cells - i.e., dendritic cells (DCs). Here we present the effects of varying pore size of poly (2-hydroxyethyl methacrylate) (pHEMA) and poly(dimethylsiloxane) (PDMS, silicone) scaffolds on the maturation and in vivo enrichment of DCs. We employ a precision templating method to make 3-D porous polymer scaffolds with uniformly defined and adjustable architecture. Hydrophilic pHEMA and hydrophobic PDMS scaffolds were fabricated in 3 pore sizes (20 μm, 40 μm, 90 μm) to quantify scaffold pore size effects on DCs activation/maturation in vitro and in vivo. In vitro results showed that both pHEMA and PDMS scaffolds could promote maturation in the DC cell line, JAWSII, that resembled lipopolysaccharide (LPS)-activated/matured DCs (mDCs). Scaffolds with smaller pore sizes correlate with higher DC maturation, regardless of the polymer used. In vivo, when implanted subcutaneously in C57BL/6J mice, scaffolds with smaller pore sizes also demonstrated more DCs recruitment and more sustained activation. Without the use of DC chemo-attractants or chemical adjuvants, our results suggested that DC maturation and scaffold infiltration profile can be modulated by simply altering the pore size of the scaffolds. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-27T06:59:39.351253-05:
      DOI: 10.1002/bit.26532
  • Methanol independent induction in Pichia pastoris by simple derepressed
           overexpression of single transcription factors
    • Authors: Thomas Vogl; Lukas Sturmberger, Pia C. Fauland, Patrick Hyden, Jasmin Elgin Fischer, Christian Schmid, Gerhard G. Thallinger, Martina Geier, Anton Glieder
      Abstract: Carbon source regulated promoters are well-studied standard tools for controlling gene expression. Acquiring control over the natural regulation of promoters is important for metabolic engineering and synthetic biology applications. In the commonly used protein production host Komagataella phaffii (Pichia pastoris), methanol-inducible promoters are used because of their tight regulation and exceptional strength. Yet, induction with toxic and flammable methanol can be a considerable safety risk and cannot be applied in many existing fermentation plants.Here we studied new regulatory circuits based on the most frequently used alcohol oxidase 1 promoter (PAOX1), which is tightly repressed in presence of repressing carbon sources and strongly induced by methanol. We compared different overexpression strategies for putative carbon source dependent regulators identified by a homology search in related yeasts and previously published literature in order to convert existing methanol dependent expression strains into methanol free systems. While constitutive overexpression showed only marginal or detrimental effects, derepressed expression (activated when the repressing carbon source is depleted) showed that three transcription factors (TFs) are single handedly suitable to strongly activate PAOX1 in P. pastoris without relying on any specifically engineered host strains. Transcriptome analyses demonstrated that Mxr1, Mit1 and Prm1 regulate partly overlapping and unique sets of genes. Derepressed overexpression of a single TF was sufficient to retrofit existing PAOX1 based expression strains into glucose/glycerol regulated, methanol-free systems. Given the wide applicability of carbon source regulated promoters, the simplicity and low cost of controlling carbon source feed rates in large scale bioreactors, similar approaches as in P. pastoris may also be useful in other organisms. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-27T06:59:22.822733-05:
      DOI: 10.1002/bit.26529
  • Perfusion applied to a 3D model of bone metastasis results in uniformly
           dispersed mechanical stimuli
    • Authors: Boyuan Liu; Suyue Han, Brandon P. Hedrick, Yahya Modarres-Sadeghi, Maureen E. Lynch
      Abstract: Breast cancer most frequently metastasizes to the skeleton. Bone metastatic cancer is incurable and induces wide-spread bone osteolysis, resulting in significant patient morbidity and mortality. Mechanical cues in the skeleton are an important microenvironmental parameter that modulate tumor formation, osteolysis, and tumor cell-bone cell signaling, but which mechanical signals are the most beneficial and the corresponding molecular mechanisms are unknown. We focused on interstitial fluid flow based on its well-known role in bone remodeling and in primary breast cancer. We created a full-scale, microCT-based computational model of a 3D model of bone metastasis undergoing applied perfusion to predict the internal mechanical environment during in vitro experimentation. Applied perfusion resulted in uniformly dispersed, heterogeneous fluid velocities and wall shear stresses throughout the scaffold's interior. The distributions of fluid velocity and wall shear stress did not change within model sub-domains of varying diameter and location. Additionally, the magnitude of these stimuli is within the range of anabolic mechanical signals in the skeleton, verifying that our 3D model reflects previous in vivo studies using anabolic mechanical loading in the context of bone metastasis. Our results indicate that local populations of cells throughout the scaffold would experience similar mechanical microenvironments. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-26T10:55:34.741416-05:
      DOI: 10.1002/bit.26524
  • Computer-Assisted Engineering of Hyperstable Fibroblast Growth Factor 2
    • Authors: Pavel Dvorak; David Bednar, Pavel Vanacek, Lukas Balek, Livia Eiselleova, Veronika Stepankova, Eva Sebestova, Michaela Kunova Bosakova, Zaneta Konecna, Stanislav Mazurenko, Antonin Kunka, Tereza Vanova, Karolina Zoufalova, Radka Chaloupkova, Jan Brezovsky, Pavel Krejci, Zbynek Prokop, Petr Dvorak, Jiri Damborsky
      Abstract: Fibroblast growth factors (FGFs) serve numerous regulatory functions in complex organisms, and their corresponding therapeutic potential is of growing interest to academics and industrial researchers alike. However, applications of these proteins are limited due to their low stability. Here we tackle this problem using a generalizable computer-assisted protein engineering strategy to create a unique modified FGF2 with nine mutations displaying unprecedented stability and uncompromised biological function. The data from the characterization of stabilized FGF2 showed a remarkable prediction potential of in silico methods and provided insight into the unfolding mechanism of the protein. The molecule holds a considerable promise for stem cell research and medical or pharmaceutical applications. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-26T10:55:29.72774-05:0
      DOI: 10.1002/bit.26531
  • Towards industrial production of isoprenoids in Escherichia coli: lessons
           learned from CRISPR-Cas9 based optimization of a chromosomally integrated
           mevalonate pathway
    • Authors: Jorge Alonso-Gutierrez; Daisuke Koma, Qijun Hu, Yuchen Yang, Leanne Jade G. Chan, Christopher J. Petzold, Paul D. Adams, Claudia E. Vickers, Lars K. Nielsen, Jay D. Keasling, Taek Soon Lee
      Abstract: Escherichia coli has been the organism of choice for the production of different chemicals by engineering native and heterologous pathways. In the present study, we simultaneously address some of the main issues associated with E. coli as an industrial platform for isoprenoids, including an inability to grow on sucrose, a lack of endogenous control over toxic mevalonate (MVA) pathway intermediates, and the limited pathway engineering into the chromosome. As a proof of concept, we generated an E. coli DH1 strain able to produce the isoprenoid bisabolene from sucrose by integrating the cscAKB operon into the chromosome and by expressing a heterologous MVA pathway under stress-responsive control. Production levels dropped dramatically relative to plasmid-mediated expression when the entire pathway was integrated into the chromosome. In order to optimize the chromosomally integrated MVA pathway, we established a CRISPR-Cas9 system to rapidly and systematically replace promoter sequences. This strategy led to higher pathway expression and a 5-fold improvement in bisabolene production. More interestingly, we analyzed proteomics data sets to understand and address some of the challenges associated with metabolic engineering of the chromosomally integrated pathway. This report shows that integrating plasmid-optimized operons into the genome and making them work optimally is not a straightforward task and any poor engineering choices on the chromosome may lead to cell death rather than just resulting in low titers. Based on these results, we also propose directions for chromosomal metabolic engineering. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-26T10:55:25.74181-05:0
      DOI: 10.1002/bit.26530
  • Central pathway engineering for enhanced succinate biosynthesis from
           acetate in Escherichia coli
    • Authors: Bing Huang; Hao Yang, Guochen Fang, Xing Zhang, Hui Wu, Zhimin Li, Qin Ye
      Abstract: Acetate, a non-food based substrate obtained from multiple biological and chemical ways, is now being paid great attention in bio-manufacturing and have a strong potential to compete with sugar-based carbon source. In this study, acetate can be efficiently converted to succinate by engineered Escherichia coli strains via the combination of several metabolic engineering strategies, including reducing OAA decarboxylation, engineering TCA cycle, enhancement of acetate assimilation pathway and increasing aerobic ATP supply through cofactor engineering. The engineered strain HB03(pTrc99a-gltA, pBAD33-Trc-fdh) accumulated 30.9 mM of succinate in 72 h and the yield reached the maximum theoretical yield (∼0.50 mol/mol). In the resting-cell experiments, the yield of succinate in HB03(pTrc99a-gltA) and HB03(pTrc99a-gltA, pBAD33-Trc-fdh) dropped dramatically, although the productivity of succinate increased due to the high cell density. Further deletion of icdA, formed HB04(pTrc99a-gltA) and HB04(pTrc99a-gltA, pBAD33-Trc-fdh), increased the yield of succinate in the resting-cell experiments. The highest concentration of succinate achieved 194 mM and the yield reached 0.44 mol/mol in 16 hours by HB04(pTrc99a-gltA, pBAD33-Trc-fdh). The results showed the metabolically engineered E. coli strains have great potential to produce succinate from acetate. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-26T10:55:21.992757-05:
      DOI: 10.1002/bit.26528
  • Manipulation of the sodium-potassium ratio as a lever for controlling cell
           growth and improving cell specific productivity in perfusion CHO cell
    • Authors: Samantha Wang; Alexandria Lee-Goldman, Janani Ravikrishnan, Lili Zheng, Henry Lin
      Abstract: Perfusion processes typically require removal of a continuous or semi-continuous volume of cell culture in order to maintain a desired target cell density. For fast growing cell lines, the product loss from this stream can be upwards of 35%, significantly reducing the overall process yield. As volume removed is directly proportional to cell growth, the ability to modulate growth during perfusion cell culture production thus becomes crucial. Leveraging existing media components to achieve such control without introducing additional supplements is most desirable because it decreases process complexity and eliminates safety and clearance concerns. Here, the impact of extracellular concentrations of sodium (Na) and potassium (K) on cell growth and productivity is explored. High throughput small-scale models of perfusion revealed Na:K ratios below 1 can significantly suppress cell growth by inducing cell cycle arrest in the G0/1 phase. A concomitant increase in cell specific productivity was also observed, reaching as high as 115 pg/cell/day for one cell line studied. Multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrated similar responses to lower Na:K media, indicating the universal applicability of such an approach. Product quality attributes were also assessed and revealed that effects were cell line specific, and can be acceptable or manageable depending on the phase of the drug development. Drastically altering Na and K levels in perfusion media as a lever to impact cell growth and productivity is proposed. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-26T10:55:19.712418-05:
      DOI: 10.1002/bit.26527
  • Botulinum hemagglutinin–mediated in situ break–up of human induced
           pluripotent stem cell aggregates for high–density suspension culture
    • Authors: Suman Chandra Nath; Tokura Tomohiro, Mee-Hae Kim, Masahiro Kino-oka
      Abstract: Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank. Although suspension culture yields high cell numbers, there remain unresolved challenges for obtaining high–density of hiPSCs because large size aggregates exhibit low growth rates. Here, we established a simple method for hiPSC aggregate break–up using botulinum hemagglutinin (HA), which specifically bound with E–cadherin and disrupted cell–cell connections in hiPSC aggregates. HA showed temporary activity for disrupting the E–cadherin–mediated cell–cell connections to facilitate the break-up of aggregates into small sizes only 9 h after HA addition. The transportation of HA into the aggregates was mediated by transcellular and paracellular way after HA addition to the culture medium. hiPSC aggregates broken up by HA showed a higher number of live cells, higher cell density, and higher expansion fold compared to those of aggregates dissociated with enzymatic digestion. Moreover, a maximum cell density of 4.5 ±0.2 × 106 cells mL−1 was obtained by aggregate break–up into small ones, which was 3 times higher than that with the conventional culture without aggregate break–up. Therefore, the temporary activity of HA for disrupting E–cadherin–mediated cell–cell connection was key to establishing a simple in situ method for hiPSC aggregate break–up in bioreactors, leading to high cell density in suspension culture. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-26T10:50:27.045394-05:
      DOI: 10.1002/bit.26526
  • Engineering Tunable Biosensors for Monitoring Putrescine in Escherichia
    • Authors: Xue-Feng Chen; Xiao-Xia Xia, Sang Yup Lee, Zhi-Gang Qian
      Abstract: Biosensors can be a powerful tool for real-time monitoring of specific small molecules and for precise control of gene expression in biological systems. Thus, biosensors have attracted much attention for monitoring increasing number of molecules. However, strategies to tune the properties of biosensors remain less explored, which might restrict their wide applicability. Here we report the development of tunable biosensors for monitoring putrescine, an important member of biological polyamines, in Escherichia coli. The native putrescine-responsive PuuR repressor protein was employed as a sensing component, and its cognate operator was installed in engineered promoters to control the expression of downstream green fluorescent protein (GFP)mut3 as a reporter protein. The engineered biosensors were specific for putrescine, and the response time could be modulated by altering growth medium of the biosensor strains. In addition, the response dynamics and detection ranges of the biosensors can be tuned at the genetic level by modulation of PuuR expression, and by manipulation of the chromosomal genes involved in putrescine biosynthesis. To demonstrate utility of the biosensors, we were able to monitor the changes of endogenous putrescine levels caused by genetic manipulations. Furthermore, a link between the excretory putrescine titer and intracellular GFP fluorescence was established for an E. coli strain that was engineered for improved putrescine biosynthesis and excretion. This study provides a strategy for engineering synthetic biosensor circuit for monitoring and tuning the dynamics in sensing putrescine, which can be generally applicable for monitoring other chemicals through taking a similar approach in circuit design. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-18T06:04:46.805533-05:
      DOI: 10.1002/bit.26521
  • Hypoxia and Transforming Growth Factor-Beta1 Pathway Activation Promote
           Chinese Hamster Ovary Cell Aggregation
    • Authors: Yueming Qian; Matthew S. Rehmann, Nan-Xin Qian, Aiqing He, Michael C. Borys, Paul S. Kayne, Zheng Jian Li
      Abstract: Suspension cultivation is the preferred mode of operation for the large-scale production of many biologics. Chinese Hamster Ovary (CHO) cells are anchorage-dependent in origin, but they have been widely adapted to suspension culture. In suspension culture, formation of CHO cell aggregates is a common phenomenon and compromises cell culture performance in multiple ways. To better understand the underlying mechanisms that regulate cell aggregation, we utilized CHO-specific transcriptome profiling as a screening tool and demonstrated that many genes encoding extracellular matrix (ECM) proteins were upregulated in the cultures with increased cell aggregation. Significantly, hypoxia was identified to be a cause for promoting CHO cell aggregation, and transforming growth factor beta1 (TGFβ1) pathway activation served as an intermediate step mediating this biological cascade. These transcriptomics findings were confirmed by cell culture experiments, and it was further demonstrated that adding recombinant TGFβ1 to the culture significantly increased ECM protein fibronectin expression and cell aggregation. The results of this study emphasize the importance of adequate mixing and oxygen supply for suspension cultures from a new angle, and regulating the TGFβ1 pathway is proposed as a new strategy for mitigating cell aggregation to improve cell culture performance. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-18T06:04:32.750828-05:
      DOI: 10.1002/bit.26520
  • 3D stromal tissue equivalent affects intestinal epithelium morphogenesis
           in vitro
    • Authors: Vincenza De Gregorio; Giorgia Imparato, Francesco Urciuolo, Paolo A. Netti
      Abstract: Current in vitro models of human intestine commonly fail to mimic the complex intestinal functions and features required for drug development and disease research. Here we deeply investigate the interaction existing between epithelium and the underneath stroma, and its role in the epithelium morphogenesis. We cultured human intestinal subepithelial myofibroblasts (ISEMFs) in two different 3D configurations: 3D-collagen gel equivalent (3D-CGE) and 3D cell-synthetized stromal equivalent (3D-CSSE). The 3D-CGEs were obtained by means of the traditional collagen-based cell technique and the 3D-CSSE were obtained by bottom-up tissue engineering strategy. The biophysical properties of both 3D models with regard to cell growth and composition (via histological analysis, immunofluorescence and multiphoton imaging) were assessed. Then, human colorectal adenocarcinoma cell line (CaCo-2) was cultured on both the 3D constructs in order to produce the intestinal model. We identified higher levels of matrix-associated proteins from ISEMFs cultured in 3D-CSSE compared to 3D-CGE. Furthermore, multiphoton investigation revealed differences in the collagen network architecture in both models. At last, the more physiologically relevant stromal environment of the 3D-CSSE drove the CaCo-2 cell differentiation towards the four different type of intestinal epithelial cells (absorptive, mucus-secretory, enteroendocrine and Paneth) phenotype and promotes, in contrast to the 3D-CGE, the production of the basement membrane. Taken together, these results highlight a fundamental role of the 3D stromal environment in addressing a correct epithelium morphogenesis as well as epithelial-stromal interface establishment. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-18T06:04:30.803331-05:
      DOI: 10.1002/bit.26522
  • Engineered cell migration to lesions linked to autoimmune disease
    • Authors: Abdullah A. Mosabbir; Anam Qudrat, Kevin Truong
      Abstract: The damaging and degenerative effects in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and Crohn's disease often manifests as the formation of lesions that feature a high local concentration of granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF along with other pro-inflammatory factors form a positive feedback loop that ultimately perpetuate the lesions. Hence, to engineer chemotaxis to GM-CSF, we created a new chimeric GM-CSF receptor alpha subunit (GMRchi) that was coupled with a previously engineered Ca2+-activated RhoA. When these proteins were expressed in mammalian cells, it allowed migration to chemical and cellular sources of GM-CSF. As a possible therapeutic intervention, we further implemented the mechanism of cell-cell membrane fusion and subsequent death. Since the microenvironment of lesions is more than just GM-CSF secretion, the further ability to recognize a combination of other features such as tissue markers will be needed for greater specificity. Nonetheless, this work represents a first step to enable cell-based therapy of autoimmune lesions. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-18T06:04:23.842507-05:
      DOI: 10.1002/bit.26523
  • Microfluidic platforms for the study of neuronal injury in vitro
    • Authors: Anil B. Shrirao; Frank H. Kung, Anton Omelchenko, Rene S. Schloss, Nada N. Boustany, Jeffrey D. Zahn, Martin L. Yarmush, Bonnie L. Firestein
      Abstract: Traumatic brain injury (TBI) affects 5.3 million people in the United States, and there are 12,500 new cases of spinal cord injury (SCI) every year. There is yet a significant need for in vitro models of TBI and SCI in order to understand the biological mechanisms underlying central nervous system (CNS) injury and to identify and test therapeutics to aid in recovery from neuronal injuries. While TBI or SCI studies have been aided with traditional in vivo and in vitro models, the innate limitations in specificity of injury, isolation of neuronal regions, and reproducibility of these models can decrease their usefulness in examining the neurobiology of injury. Microfluidic devices provide several advantages over traditional methods by allowing researchers to 1) examine the effect of injury on specific neural components, 2) fluidically isolate neuronal regions to examine specific effects on subcellular components, and 3) reproducibly create a variety of injuries to model TBI and SCI. These microfluidic devices are adaptable for modeling a wide range of injuries, and in this review, we will examine different methodologies and models recently utilized to examine neuronal injury. Specifically, we will examine vacuum-assisted axotomy, physical injury, chemical injury, and laser-based axotomy. Finally, we will discuss the benefits and downsides to each type of injury model and discuss how researchers can use these parameters to pick a particular microfluidic device to model CNS injury. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-18T05:59:02.921522-05:
      DOI: 10.1002/bit.26519
  • Fabrication and evaluation of 3D printed BCP scaffolds reinforced with
           ZrO2 for bone tissue applications
    • Authors: Min-Woo Sa; Bao-Ngoc B. Nguyen, Rebecca A. Moriarty, Timur Kamalitdinov, John P. Fisher, Jong Young Kim
      Abstract: Fused deposition modeling (FDM) is a promising 3D printing and manufacturing step to create well interconnected porous scaffold designs from the computer-aided design (CAD) models for the next generation of bone scaffolds. The purpose of this study was to fabricate and evaluate a new biphasic calcium phosphate (BCP) scaffold reinforced with zirconia (ZrO2) by a FDM system for bone tissue engineering. The 3D slurry foams with blending agents were successfully fabricated by a FDM system. Blending materials were then removed after the sintering process at high temperature to obtain a targeted BCP/ZrO2 scaffold with the desired pore characteristics, porosity, and dimension. Morphology of the sintered scaffold was investigated with SEM/EDS mapping. A cell proliferation test was carried out and evaluated with osteosarcoma MG-63 cells. Mechanical testing and cell proliferation evaluation demonstrated that 90% BCP and 10% ZrO2 scaffold had a significant effect on the mechanical properties maintaining a structure compared that of only 100% BCP with no ZrO2. Additionally, differentiation studies of human mesenchymal stem cells (hMSCs) on BCP/ZrO2 scaffolds in static and dynamic culture conditions showed increased expression of bone morphogenic protein-2 (BMP-2) when cultured on BCP/ZrO2 scaffolds under dynamic conditions compared to on BCP control scaffolds. The manufacturing of BCP/ZrO2 scaffolds through this innovative technique of a FDM may provide applications for various types of tissue regeneration, including bone and cartilage. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-14T09:31:12.600579-05:
      DOI: 10.1002/bit.26514
  • Metabolic characterization of anaerobic fungi provides a path forward for
           bioprocessing of crude lignocellulose
    • Authors: John K. Henske; St. Elmo Wilken, Kevin V. Solomon, Chuck R. Smallwood, Vaithiyalingam Shutthanandan, James E. Evans, Michael K. Theodorou, Michelle A. O'Malley
      Abstract: The conversion of lignocellulose-rich biomass to bio-based chemicals and higher order fuels remains a grand challenge, as single-microbe approaches often cannot drive both deconstruction and chemical production steps. In contrast, consortia based bioprocessing leverages the strengths of different microbes to distribute metabolic loads and achieve process synergy, product diversity, and bolster yields. Here, we describe a biphasic fermentation scheme that combines the lignocellulolytic action of anaerobic fungi isolated from large herbivores with domesticated microbes for bioproduction. When grown in batch culture, anaerobic fungi release excess sugars from both cellulose and crude biomass due to a wealth of highly expressed carbohydrate active enzymes (CAZymes), converting as much as 49% of cellulose to free glucose. This sugar-rich hydrolysate readily supports growth of S. cerevisiae, which can be engineered to produce a range of value-added chemicals. Further, construction of metabolic pathways from transcriptomic data reveals that anaerobic fungi do not catabolize all sugars that their enzymes hydrolyze from biomass, leaving other carbohydrates such as galactose, arabinose, and mannose available as nutritional links to other microbes in their consortium. Although basal expression of CAZymes in anaerobic fungi is high, it is drastically amplified by cellobiose breakout products encountered during biomass hydrolysis. Overall, these results suggest that anaerobic fungi provide a nutritional benefit to the rumen microbiome, which can be harnessed to design synthetic microbial communities that compartmentalize biomass degradation and bioproduct formation. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-14T09:30:58.682381-05:
      DOI: 10.1002/bit.26515
  • Advances in applications of rhamnolipids biosurfactant in environmental
           remediation: A review
    • Authors: Guansheng Liu; Hua Zhong, Xin Yang, Yang Liu, Binbin Shao, Zhifeng Liu
      Abstract: The objective of this review is to provide a comprehensive overview of the advances in the applications of rhamnolipids biosurfactants in soil and ground water remediation for removal of petroleum hydrocarbon and heavy metal contaminants. The properties of rhamnolipids associated with the contaminant removal, i.e. solubilization, emulsification, dispersion, foaming, wetting, complexation, and the ability to modify bacterial cell surface properties, were reviewed in the first place. Then current remediation technologies with integration of rhamnolipid were summarized, and the effects and mechanisms for rhamnolipid to facilitate contaminant removal for these technologies were discussed. Finally rhamnolipid-based methods for remediation of the sites co-contaminated by petroleum hydrocarbons and heavy metals were presented and discussed. The review is expected to enhance our understanding on environmental aspects of rhamnolipid and provide some important information to guide the extending use of this fascinating chemical in remediation applications. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-14T09:27:14.621611-05:
      DOI: 10.1002/bit.26517
  • Engineering and cytosolic delivery of a native regulatory protein and its
           variants for modulation of ERK2 signaling pathway
    • Authors: Jeong-Hyun Ryou; Yoo-Kyoung Sohn, Dong-Gun Kim, Hyun-Ho Kyeong, Hak-Sung Kim
      Abstract: The modulation of a cell signaling process using a molecular binder followed by an analysis of the cellular response is crucial for understanding its role in the cellular function and developing pharmaceuticals. Herein, we present the modulation of the ERK2-mediated signaling pathway through the cytosolic delivery of a native regulatory protein for ERK2, i.e, PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa), and its engineered variants using a bacterial toxin-based delivery system. Based on biochemical and structural analyses, PEA-15 variants with different phosphorylation sites and a high affinity for ERK2 were designed. Semi-rational approach led to about an 830-fold increase in the binding affinity of PEA-15, resulting in more effective modulation of the ERK2-mediated signaling. Our approach enabled an understanding of the cellular function of the ERK2-mediated signaling process and the effect of PEA-15 phosphorylation on its action as an ERK2 blocker. We demonstrated the utility and potential of our approach by showing an efficient cytosolic delivery of these PEA-15 variants and the effective suppression of cell proliferation through the inhibition of the ERK2 function. The present approach can be used broadly for modulating the cell signaling processes and understanding their roles in cellular function, as well as for the development of therapeutics. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-14T09:26:51.364109-05:
      DOI: 10.1002/bit.26516
  • Thermoactivation of a cellobiohydrolase
    • Authors: Peter Westh; Kim Borch, Trine Sørensen, Radina Tokin, Jeppe Kari, Silke Badino, A. Mafalda Cavaleiro, Nanna Røjel, Stefan Christensen, Cynthia S. Vesterager, Corinna Schiano-di-Cola
      Abstract: We have measured activity and substrate affinity of the thermostable cellobiohydrolase, Cel7A, from Rasamsonia emersonii over a broad range of temperatures. For the wild type enzyme, which does not have a Carbohydrate Binding Module (CBM), higher temperature only led to moderately increased activity against cellulose, and we ascribed this to a pronounced, temperature induced desorption of enzyme from the substrate surface. We also tested a “high affinity” variant of R. emersonii Cel7A with a linker and CBM from a related enzyme. At room temperature, the activity of the variant was similar to the wild type, but the variant was more accelerated by temperature and about 2-fold faster around 70°C. This better thermoactivation of the high-affinity variant could not be linked to differences in stability or the catalytic process, but coincided with less desorption as temperature increased. Based on these observations and earlier reports on moderate thermoactivation of cellulases, we suggest that better cellulolytic activity at industrially relevant temperatures may be attained by engineering improved substrate affinity into enzymes that already possess good thermostability. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-14T09:20:32.74331-05:0
      DOI: 10.1002/bit.26513
  • Modeling Electron Competition among Nitrogen Oxides Reduction and N2O
           Accumulation in Hydrogenotrophic Denitrification
    • Authors: Yiwen Liu; Huu Hao Ngo, Wenshan Guo, Lai Peng, Xueming Chen, Dongbo Wang, Yuting Pan, Bing-Jie Ni
      Abstract: Hydrogenotrophic denitrification is a novel and sustainable process for nitrogen removal, which utilizes hydrogen as electron donor and carbon dioxide as carbon source. Recent studies have shown that nitrous oxide (N2O), a highly undesirable intermediate and potent greenhouse gas, can accumulate during this process. In this work, a new mathematical model is developed to describe nitrogen oxides dynamics, especially N2O, during hydrogenotrophic denitrification for the first time. The model describes electron competition among the four steps of hydrogenotrophic denitrification through decoupling hydrogen oxidation and nitrogen reduction processes using electron carriers, in contrast to the existing models that couple these two processes and also do not consider N2O accumulation. The developed model satisfactorily describes experimental data on nitrogen oxides dynamics obtained from two independent hydrogenotrophic denitrifying cultures under various hydrogen and nitrogen oxides supplying conditions, suggesting the validity and applicability of the model. The results indicated that N2O accumulation would not be intensified under hydrogen limiting conditions, due to the higher electron competition capacity of N2O reduction in comparison to nitrate and nitrite reduction during hydrogenotrophic denitrification. The model is expected to enhance our understanding of the process during hydrogenotrophic denitrification and the ability to predict N2O accumulation. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-14T09:20:25.556115-05:
      DOI: 10.1002/bit.26512
  • Maximizing neotissue growth kinetics in a perfusion bioreactor: an in
           silico strategy using model reduction and Bayesian optimization
    • Authors: Mohammad Mehrian; Yann Guyot, Ioannis Papantoniou, Simon Olofsson, Maarten Sonnaert, Ruth Misener, Liesbet Geris
      Abstract: In regenerative medicine, computer models describing bioreactor processes can assist in designing optimal process conditions leading to robust and economically viable products. In this study, we started from a (3D) mechanistic model describing the growth of neotissue, comprised of cells and extracellular matrix, in a perfusion bioreactor set-up influenced by the scaffold geometry, flow-induced shear stress and a number of metabolic factors. Subsequently, we applied model reduction by reformulating the problem from a set of partial differential equations into a set of ordinary differential equations. Comparing the reduced model results to the mechanistic model results and to dedicated experimental results assesses the reduction step quality. The obtained homogenized model is 105 fold faster than the 3D version, allowing the application of rigorous optimization techniques. Bayesian optimization was applied to find the medium refreshment regime in terms of frequency and percentage of medium replaced that would maximize neotissue growth kinetics during 21 days of culture. The simulation results indicated that maximum neotissue growth will occur for a high frequency and medium replacement percentage, a finding that is corroborated by reports in the literature. This study demonstrates an in silico strategy for bioprocess optimization paying particular attention to the reduction of the associated computational cost. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-04T06:31:19.746056-05:
      DOI: 10.1002/bit.26500
  • Paramagnetism in Bacillus spores: opportunities for novel biotechnological
    • Authors: Ke Xu Zhou; Adrian Ionescu, Eamon Wan, Yeuk Nam Ho, Crispin H. W. Barnes, Graham Christie, D. Ian Wilson
      Abstract: Spores of Bacillus megaterium, B. cereus and B. subtilis were found to exhibit intrinsic paramagnetic properties as a result of the accumulation of manganese ions. All three Bacillus species displayed strong yet distinctive magnetic properties arising from differences in manganese quantity and valency. Manganese ions were found to accumulate both within the spore core as well as being associated with the surface of the spore. Bacillus megaterium spores accumulated up to 1 wt. % manganese (II) within, with a further 0.6 wt. % adsorbed onto the surface. At room temperature, Bacillus spores possess average magnetic susceptibilities in the range of 10−6 to 10−5. Three spore-related biotechnological applications - magnetic sensing, magnetic separation and metal ion adsorption - were assessed subsequently, with the latter two considered as having the most potential for development. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-04T06:30:47.134408-05:
      DOI: 10.1002/bit.26501
  • Vitamin B12 association with mAbs: Mechanism and Potential Mitigation
    • Authors: Cheng Du; Robert Martin, Yunping Huang, Ameya Borwankar, Zhijun Tan, Jay West, Nripen Singh, Michael Borys, Sanchayita Ghose, Richard Ludwig, Li Tao, Zheng Jian Li
      Abstract: Process control for manufacturing biologics is critical for ensuring product quality, safety and lot to lot consistency of therapeutic proteins. In this study, we investigated the root cause of the pink coloration observed for various in-process pools and drug substances in the antibody manufacturing process. Vitamin B12 is covalently bound to mAbs via a cobalt-sulfur coordinate bond via the cysteine residues. The vitamin B12 was identified to attach to an IgG4 molecule at cysteine residues on light chain (Cys-214), and heavy chain (Cys-134, Cys-321, Cys-367 and Cys-425). Prior to attachment to mAbs, the vitamin B12 needs to be in its active form of hydroxocobalamin. During culture media preparation, storage and cell culture processing, cyanocobalamin, the chemical form of vitamin B12 added to media, is converted to hydroxocobalamin by white fluorescence light (about 50% degradation in 11–14 days at room temperature and with room light intensity about 500–1,000 lux) and by short-wavelength visible light (400–550 nm). However, cyanocobalamin is stable under red light (wavelength > 600 nm) exposure and does not convert to hydroxocobalamin. Our findings suggests that the intensity of pink color depends on concentrations of both free sulfhydryl groups on reduced mAb and hydroxocobalamin, the active form of vitamin B12. Both reactants are necessary and neither one of them is sufficient to generate pink color, therefore process control strategy can consider limiting either one or both factors. A process control strategy to install red light (wavelength > 600 nm) in culture media preparation, storage and culture processing areas is proposed to provide safe light for biologics and to prevent light-induced color variations in final products. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-02T15:25:30.235261-05:
      DOI: 10.1002/bit.26511
  • Growth modeling of the green microalga Scenedesmus obliquus in a hybrid
           photobioreactor as a practical tool to understand both physical and
           biochemical phenomena in play during algae cultivation
    • Authors: Deise Parolo Tramontin; Pablo Diego Gressler, Leonardo Rubi Rörig, Roberto Bianchini Derner, Jurandir Pereira Filho, Claudemir Marcos Radetski, Marintho Bastos Quadri
      Abstract: In recent years, numerous studies have justified the use of microalgae as a sustainable alternative for the generation of different types of fuels, food supplementation and cosmetics, as well as bioremediation processes. To improve the cost/benefit ratio of microalgae mass production, many culture systems have been built and upgraded. Mathematical modeling the growth of different species in different systems has become an efficient and practical tool to understand both physical and biochemical phenomena in play during algae cultivation. In addition, growth modeling can guide design changes that lead to process optimization. In the present work, growth of the green microalga Scenedesmus obliquus was modeled in a hybrid photobioreactor that combines the characteristics of tubular photobioreactors (TPB) with thin-layer cascades (TLC). The system showed productivity greater than 8.0 g m−2 day−1 (dry mass) for CO2-fed cultures, and the model proved to be an accurate representation of experimental data with R2 greater than 0.7 for all cases under variable conditions of temperature and irradiance to determine subsystem efficiency. Growth modeling also allowed growth prediction relative to the operating conditions of TLC, making it useful for estimating the system given other irradiance and temperature conditions, as well as other microalgae species. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-02T15:25:27.626691-05:
      DOI: 10.1002/bit.26510
  • Production of cellulosic organic acids via synthetic fungal consortia
    • Authors: Scott A. Scholz; Ian Graves, Jeremy J. Minty, Xiaoxia Nina Lin
      Abstract: Consolidated bioprocessing is a potential breakthrough technology for reducing costs of biochemical production from lignocellulosic biomass. Production of cellulase enzymes, saccharification of lignocellulose and conversion of the resulting sugars into a chemical of interest occur simultaneously within a single bioreactor. In this study, synthetic fungal consortia composed of the cellulolytic fungus Trichoderma reesei and the production specialist Rhizopus delemar demonstrated conversion of microcrystalline cellulose (MCC) and alkaline pre-treated corn stover to fumaric acid in a fully consolidated manner without addition of cellulase enzymes or expensive supplements such as yeast extract. A titer of 6.87 g/L of fumaric acid, representing 0.17 w/w yield, were produced from 40 g/L MCC with a productivity of 31.8 mg/L/h. In addition, lactic acid was produced from MCC using a fungal consortium with Rhizopus oryzae as the production specialist. These results are proof-of-concept demonstration of engineering synthetic microbial consortia for CBP production of naturally occurring biomolecules. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-02T15:20:42.25713-05:0
      DOI: 10.1002/bit.26509
  • Production of itaconic acid from acetate by engineering acid-tolerant
           Escherichia coli W
    • Authors: Myung Hyun Noh; Hyun Gyu Lim, Sung Hwa Woo, Jinyi Song, Gyoo Yeol Jung
      Abstract: Utilization of abundant and cheap carbon sources can effectively reduce the production cost and enhance the economic feasibility. Acetate is a promising carbon source to achieve cost-effective microbial processes. In this study, we engineered an Escherichia coli strain to produce itaconic acid from acetate. Since acetate is known to inhibit cell growth, we initially screened for a strain with a high tolerance to 10 g/L of acetate in the medium, and the W strain was selected as the host. Subsequently, the WC strain was obtained by overexpression of cad (encoding cis-aconitate decarboxylase) using a synthetic promoter and 5' UTR. However, the WC strain produced only 0.13 g/L itaconic acid because of low acetate uptake. To improve the production, the acetate assimilating pathway and glyoxylate shunt pathway were amplified by overexpression of pathway genes as well as its deregulation. The resulting strain, WCIAG4 produced 3.57 g/L itaconic acid (16.1% of theoretical maximum yield) after 88 h of fermentation with rapid acetate assimilation. These efforts support that acetate can be a potential feedstock for biochemical production with engineered E. coli. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-02T06:45:46.275962-05:
      DOI: 10.1002/bit.26508
  • Development of a microfluidic platform for high-throughput screening of
           non-viral gene delivery vectors
    • Authors: Elisa Giupponi; Roberta Visone, Paola Occhetta, Federica Colombo, Marco Rasponi, Gabriele Candiani
      Abstract: The grail of gene delivery is the development of delivery vectors as effective and non-cytotoxic as possible. In this regard, there is an urgent need of new tools for the straightforward and quantitative assessment of transfection efficiency and cytotoxicity simultaneously. We herein reported the development and validation of an easy-to-use lab-on-chip platform to perform cell transfection assays for unbiased, high-throughput selection of more and more effective gene delivery vectors by using 2 commercially sourced lipids, Lipofectamine 2000® and FuGene® 6. A single PDMS-layer platform was endowed with i) a chaotic serial dilution generator, designed for the automatic generation of a linear lipoplex dilution (from 100% to 0% with 25% steps) independently delivered to ii) the downstream culture and transfection module consisting in 5 units, each composed of 33 serially- and fluidically-connected culture chambers for trapping small populations of ≈10 cells/chamber. In the absence of any transfectant, cells spread and duplicated up to 2 days. Besides, cells were transfected with EGFP-encoding reporter gene. The very facile visual inspection of the microdevice by means of a microscope and a semi-automated analytical method allowed pinpointing the best transfection conditions in terms of efficiency, cytotoxicity, cell doubling rates and morphological changes at once
      PubDate: 2017-12-02T06:45:44.307959-05:
      DOI: 10.1002/bit.26506
  • Constructing a Cellulosic Yeast Host with an Efficient Cellulase Cocktail
    • Authors: Jui-Jen Chang; Yu-Ju Lin, Chyi-How Lay, Caroline Thia, Yueh-Chin Wu, Yu-Han Hou, Chieh-Chen Huang, Wen-Hsiung Li
      Abstract: Cellulose is a renewable feedstock for green industry. It is therefore important to develop a technique to construct a host with a high cellulolytic efficiency to digest cellulose. In this study, we developed a convenient host-engineering technique to adjust the expression levels of heterologous genes in the host by promoter rearrangement and gene copy number adjustment. Using genes from different glycoside hydrolase (GH) families including GH2, GH3, GH5, GH6, GH7, and GH12 from Aspergillus niger, Trichoderma reesei and Neocallimastix patriciarum, we constructed a cellulolytic Kluyveromyces marxianus with 8 cellulase gene-cassettes that produced a cellulase cocktail with a high cellulolytic efficiency, leading to a significant reduction in enzyme cost in a rice straw saccharification process. Our technique can be used to design a host that can efficiently convert biomass feedstock to biofuel. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-02T06:45:25.682546-05:
      DOI: 10.1002/bit.26507
  • Investigating the Strategies for Microbial Production of Trehalose from
           Lignocellulosic Sugars
    • Authors: Yifei Wu; Jian Wang, Xiaolin Shen, Jia Wang, Zhenya Chen, Xinxiao Sun, Qipeng Yuan, Yajun Yan
      Abstract: Trehalose, a multi-functional and value-added disaccharide, can be efficiently biosynthesized from glucose by using a synergetic carbon utilization mechanism (SynCar) which coupled phosphoenolpyruvate (PEP) generation from the second carbon source with PEP-dependent phosphotransferase system (PTS) to promote non-catabolic use of glucose. Considering glucose and xylose present in large amounts in lignocellulosic sugars, we explored new strategies for conversion of both sugars into trehalose. Herein, we first attempted trehalose production from xylose directly, based on which, synergetic utilization of glucose and xylose prompted by SynCar was implemented in engineered E. coli. As the results, the final titer of trehalose reached 5.55 g/L in shake flask experiments. The conversion ratio or utilization efficiency of glucose or xylose to trehalose was around 4-fold higher than that of the original strain (YW-3). This work not only demonstrated the possibility of directly converting xylose (C5 sugar) into trehalose (C12 disaccharide), but also suggested a promising strategy for trehalose production from lignocellulosic sugars for the first time. This article is protected by copyright. All rights reserved
      PubDate: 2017-12-02T06:40:24.322478-05:
      DOI: 10.1002/bit.26505
  • A cell-free platform for rapid synthesis and testing of active
    • Authors: Jennifer A. Schoborg; Jasmine Hershewe, Jessica C. Stark, Weston Kightlinger, James E. Kath, Thapakorn Jaroentomeechai, Aravind Natarajan, Matthew P. DeLisa, Michael C. Jewett
      Abstract: Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 420 μg/mL for the single-subunit OST enzyme, 'Protein glycosylation B' (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-27T02:15:53.373577-05:
      DOI: 10.1002/bit.26502
  • N-glycan engineering of a plant-produced anti-CD20-hIL-2 immunocytokine
           significantly enhances its effector functions
    • Authors: Carla Marusic; Claudio Pioli, Szymon Stelter, Flavia Novelli, Chiara Lonoce, Elena Morrocchi, Eugenio Benvenuto, Anna Maria Salzano, Andrea Scaloni, Marcello Donini
      Abstract: Anti-CD20 recombinant antibodies are among the most promising therapeutics for the treatment of B-cell malignancies such as non-Hodgkin lymphomas. We recently demonstrated that an immunocytokine (2B8-Fc-hIL2), obtained by fusing an anti-CD20 scFv-Fc antibody derived from C2B8 mAb (rituximab) to the human interleukin 2 (hIL-2), can be efficiently produced in Nicotiana benthamiana plants. The purified immunocytokine (IC) bearing a typical plant protein N-glycosylation profile showed a CD20 binding activity comparable to that of rituximab and was efficient in eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) of human PBMC against Daudi cells, indicating its fuctional integrity. In this work, the immunocytokine devoid of the typical xylose/fucose N-glycosylation plant signature (IC-ΔXF) and the corresponding scFv-Fc- XF antibody not fused to the cytokine, were obtained in a glyco-engineered XylT/FucTΔN. benthamiana line. Purification yields from agroinfiltrated plants amounted to 20-35 mg/Kg of leaf fresh weight. When assayed for interaction with FcγRI and FcγRIIIa, IC-ΔXF exhibited significantly enhanced binding affinities if compared to the counterpart bearing the typical plant protein N-glycosylation profile (IC) and to rituximab. The glyco-engineered recombinant molecules also exhibited a strongly improved ADCC and complement-dependent cytotoxicity (CDC). Notably, our results demonstrate a reduced C1q binding of xylose/fucose carrying IC and scFv-Fc compared to versions that lack these sugar moieties. These results demonstrate that specific N-glycosylation alterations in recombinant products can dramatically affect the effector functions of the immunocytokine, resulting in an overall improvement of the biological functions and consequently of the therapeutic potential. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-27T02:10:37.769891-05:
      DOI: 10.1002/bit.26503
  • 3D aggregate culture improves metabolic maturation of human pluripotent
           stem cell derived cardiomyocytes
    • Authors: Cláudia Correia; Alexey Koshkin, Patrícia Duarte, Dongjian Hu, Madalena Carido, Maria J. Sebastião, Patrícia Gomes-Alves, David A. Elliott, Ibrahim Domian, Ana P. Teixeira, Paula M. Alves, Margarida Serra
      Abstract: Three-dimensional (3D) cultures of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) hold great promise for drug discovery, providing a better approximation to the in vivo physiology over standard two-dimensional (2D) monolayer cultures. However, the transition of CM differentiation protocols from 2D to 3D cultures is not straightforward. In this work, we relied on the aggregation of hPSC-derived cardiac progenitors and their culture under agitated conditions to generate highly pure cardiomyocyte aggregates. Whole-transcriptome analysis and 13C-metabolic flux analysis allowed to demonstrate at both molecular and fluxome levels that such 3D culture environment enhances metabolic maturation of hiPSC-CMs. When compared to 2D, 3D cultures of hiPSC-CMs displayed down-regulation of genes involved in glycolysis and lipid biosynthesis and increased expression of genes involved in OXPHOS. Accordingly, 3D cultures of hiPSC-CMs had lower fluxes through glycolysis and fatty acid synthesis and increased TCA-cycle activity. Importantly, we demonstrated that the 3D culture environment reproducibly improved both CM purity and metabolic maturation across different hPSC lines, thereby providing a robust strategy to derive enriched hPSC-CMs with metabolic features closer to that of adult CMs. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-27T02:10:35.374835-05:
      DOI: 10.1002/bit.26504
  • cGMP production and analysis of BG505 SOSIP.664, an extensively
           glycosylated, trimeric HIV-1 envelope glycoprotein vaccine candidate
    • Authors: Antu K. Dey; Albert Cupo, Gabriel Ozorowski, Vaneet K. Sharma, Anna-Janina Behrens, Eden P. Go, Thomas J. Ketas, Anila Yasmeen, Per J. Klasse, Eddy Sayeed, Heather Desaire, Max Crispin, Ian A. Wilson, Rogier W. Sanders, Thomas Hassell, Andrew Ward, John P. Moore
      Abstract: We describe the properties of BG505 SOSIP.664 HIV-1 envelope glycoprotein trimers produced under current Good Manufacturing Practice (cGMP) conditions. These proteins are the first of a new generation of native-like trimers that are the basis for many structure-guided immunogen development programs aimed at devising how to induce broadly neutralizing antibodies (bNAbs) to HIV-1 by vaccination. The successful translation of this prototype demonstrates the feasibility of producing similar immunogens on an appropriate scale and of an acceptable quality for Phase I experimental medicine clinical trials. BG505 SOSIP.664 trimers are extensively glycosylated, contain numerous disulfide bonds and require proteolytic cleavage, all properties that pose a substantial challenge to cGMP production. Our strategy involved creating a stable CHO cell line that was adapted to serum-free culture conditions to produce envelope glycoproteins. The trimers were then purified by chromatographic methods using a 2G12 bNAb affinity column and size-exclusion chromatography. The chosen procedures allowed any adventitious viruses to be cleared from the final product to the required extent of>12 log10. The final cGMP production run yielded 3.52 grams (peptidic mass) of fully purified trimers (Drug Substance) from a 200 L bioreactor, a notable yield for such a complex glycoprotein. The purified trimers were fully native-like as judged by negative-stain electron microscopy, and were stable over a multi-month period at room temperature or below and for at least one week at 50°C. Their antigenicity, disulfide bond patterns and glycan composition were consistent with trimers produced on a research laboratory scale. The methods reported here should pave the way for the cGMP production of other native-like Env glycoprotein trimers of various designs and genotypes. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-18T10:25:37.307969-05:
      DOI: 10.1002/bit.26498
  • Photoautotrophic production of macular pigment in a Chlamydomonas
           reinhardtii strain generated by using DNA-free CRISPR-Cas9 RNP-mediated
    • Authors: Kwangryul Baek; Jihyeon Yu, Jooyeon Jeong, Sang Jun Sim, Sangsu Bae, EonSeon Jin
      Abstract: Lutein and zeaxanthin are dietary carotenoids reported to be protective against age-related macular degeneration. Recently, the green alga Chlamydomonas reinhardtii has received attention as a photosynthetic cell factory, but the potential of this alga for carotenoid production has not yet been evaluated. In this study, we selected the C. reinhardtii CC-4349 strain as the best candidate among seven laboratory strains tested for carotenoid production. A knock-out mutant of the zeaxanthin epoxidase gene induced by preassembled DNA-free CRISPR-Cas9 ribonucleoproteins in the CC-4349 strain had a significantly higher zeaxanthin content (56-fold) and productivity (47-fold) than the wild type without the reduction in lutein level. Furthermore, we produced eggs fortified with lutein (2-fold) and zeaxanthin (2.2-fold) by feeding hens a diet containing the mutant. Our results clearly demonstrate the possibility of cost-effective commercial use of microalgal mutants induced by DNA-free CRISPR-Cas9 ribonucleoproteins in algal biotechnology for the production of high-value products. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-18T10:25:28.278739-05:
      DOI: 10.1002/bit.26499
  • Modulation of IgG1 Immunoeffector Function by Glycoengineering of the
           GDP-Fucose Biosynthesis Pathway
    • Authors: Ronan M. Kelly; Ronald L. Kowle, Zhirui Lian, Beth A. Strifler, Derrick R Witcher, Bhavin S. Parekh, Tongtong Wang, Christopher C. Frye
      Abstract: Cross-linking of the Fcγ receptors expressed on the surface of hematopoietic cells by IgG immune complexes triggers the activation of key immune effector mechanisms, including antibody-dependent cell mediated cytotoxicity (ADCC). A conserved N-glycan positioned at the N-terminal region of the IgG CH2 domain is critical in maintaining the quaternary structure of the molecule for Fcγ receptor engagement. The removal of a single core fucose residue from the N-glycan results in a considerable increase in affinity for FcγRIIIa leading to an enhanced receptor-mediated immunoeffector function. The enhanced potency of the molecule translates into a number of distinct advantages in the development of IgG antibodies for cancer therapy. In an effort to significantly increase the potency of an anti-CD20, IgG1 molecule, we selectively targeted the de novo GDP-fucose biosynthesis pathway of the host CHO cell line to generate>80% afucosylated IgG1 resulting in enhanced FcγRIIIa binding (13-fold) and in vitro ADCC cell-based activity (11-fold). In addition, this effective glycoengineering strategy also allowed for the utilization of the alternate GDP-fucose salvage pathway to provide a fast and efficient mechanism to manipulate the N-glycan fucosylation level to modulate IgG immune effector function. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-18T10:20:25.708995-05:
      DOI: 10.1002/bit.26496
  • Design of a Novel Continuous Flow Reactor for Low pH Viral Inactivation
    • Authors: Stephanie A. Parker; Linus Amarikwa, Kevin Vehar, Raquel Orozco, Scott Godfrey, Jon Coffman, Parviz Shamlou, Cameron L. Bardliving
      Abstract: Insufficient mixing in laminar flow reactors due to diffusion-dominated flow limits their use in applications where narrow residence time distribution (RTD) is required. The aim of this study was to design and characterize a laminar flow (Re 187.7-375.5) tubular reactor for low pH viral inactivation with enhanced radial mixing via the incorporation of curvature and flow inversions. Towards this aim, the reactor described here, Jig in a Box (JIB), was designed with a flow path consisting of alternating 270° degree turns. The design was optimized by considering the strength of secondary flows characterized by the Dean No., the corresponding secondary flow development length, and the reactor turn lengths. Comprehensive CFD analysis of the reactor centerline velocity profile, cross-sectional velocity, and secondary flow streamlines confirmed enhanced radial mixing due to secondary flows and changes in flow direction. For initial CFD and experimental studies the reactor was limited to a 16.43 m length. Pulse tracer studies for the reactor were computationally simulated and experimentally generated to determine the RTD, RTD variance, and minimum residence time for the tracer fluid elements leaving the reactor, as well as to validate the computational model. The reactor was scaled length wise to increase incubation time and it was observed that as the reactor length increases the RTD variance increases linearly and the dimensionless RTD profile becomes more symmetrical and tighter about the mean residence time. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-18T10:20:22.251287-05:
      DOI: 10.1002/bit.26497
  • A fast and simple method to estimate relative, hyphal tensile-strength of
           filamentous fungi used to assess the effect of autophagy
    • Authors: Daniela Quintanilla; Cynthia Chelius, Sirasa Iambamrung, Sidney Nelson, Donnel Thomas, Krist V. Gernaey, Mark R. Marten
      Abstract: Fungal hyphal strength is an important phenotype which can have a profound impact on bioprocess behavior. Until now, there is not an efficient method which allows its characterization. Currently available methods are very time consuming; thus, compromising their applicability in strain selection and process development. To overcome this issue, a method for fast and easy, statistically-verified quantification of relative hyphal tensile strength was developed. It involves off-line fragmentation in a high shear mixer followed by quantification of fragment size using laser diffraction. Particle size distribution (PSD) is determined, with analysis time on the order of minutes. Plots of PSD 90th percentile versus time allow estimation of the specific fragmentation rate. This novel method is demonstrated by estimating relative hyphal strength during growth in control conditions and rapamycin-induced autophagy for Aspergillus nidulans (paternal strain) and a mutant strain (ΔAnatg8) lacking an essential autophagy gene. Both strains were grown in shake flasks, and relative hyphal tensile strength was compared. The mutant strain grown in control conditions appears to be weaker than the paternal strain, suggesting that Anatg8 may play a role in other processes involving cell wall biosynthesis. Furthermore, rapamycin-induced autophagy resulted in apparently weaker cells even for the mutant strain. These findings confirm the utility of the developed method in strain selection and process development. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-14T08:10:36.031042-05:
      DOI: 10.1002/bit.26490
  • Highly active spore biocatalyst by self-assembly of co-expressed anchoring
           scaffoldin and multimeric enzyme
    • Authors: Long Chen; Megan Holmes, Elise Schaefer, Ashok Mulchandani, Xin Ge
      Abstract: We report a spore-based biocatalysis platform capable of producing and self-assembling active multimeric enzymes on a spore surface with a high loading density. This was achieved by co-expressing both a spore surface-anchoring scaffoldin protein containing multiple cohesin domains and a dockerin-tagged enzyme of interest in the mother cell compartment during Bacillus subtilis sporulation. Using this method, tetrameric β-galactosidase was successfully displayed on the spore surface with a loading density of 1.4 × 104 active enzymes per spore particle. The resulting spore biocatalysts exhibited high conversion rates of transgalactosylation in water/organic emulsions. With easy manufacture, enhanced thermostability, excellent reusability, and long-term storage stability at ambient temperature, this approach holds a great potential in a wide range of biocatalysis applications especially involving organic phases. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-13T06:55:34.129334-05:
      DOI: 10.1002/bit.26492
  • Ty1-fused Protein-body Formation for Spatial Organization of Metabolic
           Pathways in Saccharomyces cerevisiae
    • Authors: Jong Yun Han; Jae Myeong Song, Sung Hwa Seo, Chonglong Wang, Seung-Goo Lee, Hongweon Lee, Seon-Won Kim, Eui-Sung Choi
      Abstract: Metabolite production through a multistep metabolic pathway can often be increased by efficient substrate channeling created by spatial sequestration of the metabolic reactions. Here, Tya, a structural component in the Ty1 retrotransposon element that forms virus-like particles (VLPs) in Saccharomyces cerevisiae, was used to spatially organize enzymes involved in a metabolic pathway into a multi-enzyme protein body in yeast. As a proof of principle, Tya fusion to three key enzymes involved in biosynthesis of the isoprenoids farnesene and farnesol was tested to assess its potential to improve productivity. The Tya-fusion protein resulted in 3- and 4-fold increases in farnesene and farnesol production, respectively, as compared with that observed in a non-fused control. Specifically, two-phase partitioning fed-batch fermentations of S. cerevisiae ATCC200589 overexpressing Tya-fused enzymes (tHmg1, IspA, and α-farnesene synthase) yielded 930 ± 40 mg/L of farnesene after 7 days. Additionally, we observed that the Tya-fusion proteins tended to partition into particulate fractions upon 100,000g ultracentrifugation, suggesting the formation of large aggregates of protein bodies, with their particulate structure also observed by transmission electron microscopy. The dramatic increase in the biosynthetic productivity of metabolites via use of a Tya-fusion protein suggested that this approach might be useful for the creation of multi-enzyme complexes to improve metabolic engineering in yeast. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-13T06:55:23.278641-05:
      DOI: 10.1002/bit.26493
  • Glycosyltransferase Cascades Made Fit for Chemical Production: Integrated
           Biocatalytic Process for the Natural Polyphenol C-Glucoside Nothofagin
    • Authors: Katharina Schmölzer; Martin Lemmerer, Bernd Nidetzky
      Abstract: Glycosyltransferase cascades are promising tools of biocatalysis for natural product glycosylation, but their suitability for actual production remains to be shown. Here, we demonstrate at a scale of 100 g isolated product the integrated biocatalytic production of nothofagin, the natural 3'-C-β-D-glucoside of the polyphenol phloretin. A parallel reaction cascade involving coupled C-glucosyltransferase and sucrose synthase was optimized for the one-pot glucosylation of phloretin from sucrose via an UDP/UDP-glucose shuttle. Inclusion complexation with the highly water soluble 2-hydroxypropyl-β-cyclodextrin pushed the phloretin solubility to its upper practical limit (∼120 mM) and so removed the main bottleneck on an efficient synthesis of nothofagin. The biotransformation thus intensified had excellent performance metrics of 97% yield and ∼50 gproduct/L at a space-time yield of 3 g/L/h. The UDP-glucose was regenerated up to ∼220 times. A scalable downstream process for efficient recovery of nothofagin (≥95% purity; ≥65% yield) was developed. A tailored anion-exchange chromatography at pH 8.5 was used for capture and initial purification of the product. Recycling of the 2-hydroxypropyl-β-cyclodextrin would also be possible at this step. Product precipitation at a lowered pH of 6.0 and re-dissolution in acetone effectively replaced desalting by size exclusion chromatography in the final step of nothofagin purification. This study therefore reveals the potential for process intensification in the glycosylation of polyphenol acceptors by glycosyltransferase cascades. It demonstrates that, with up- and downstream processing carefully optimized and suitably interconnected, a powerful biocatalytic technology becomes available for the production of an important class of glycosides difficult to prepare otherwise. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-13T06:50:28.739968-05:
      DOI: 10.1002/bit.26491
  • Electron shuttling to ferrihydrite selects for fermentative rather than
           Fe3+-reducing biomass in xylose-fed batch reactors derived from three
           different inoculum sources
    • Authors: Jovan Popovic; Kevin T. Finneran
      Abstract: Reports suggest that ferric iron and electron shuttling molecules will select for Fe3+-reducer dominated microbial biomass. We investigated the influence of the redox mediators anthraquinone-2,6-disulfonate (AQDS) and riboflavin using xylose as the sole fermentation substrate, with or without ferric iron. Electron shuttling to insoluble ferrihydrite enhanced solventogenesis, acidogenesis, hydrogen production, and xylose consumption, relative to the cells plus xylose controls in fermentations inoculated with woodland marsh sediment, wetwood disease, or raw septic liquid, over multiple transfers in 15-day batch fermentations. 16S rRNA gene based community analyses indicated that ferrihydrite alone, and AQDS/riboflavin plus ferrihydrite, immediately shifted native heterogeneous communities to those predominantly belonging to the Clostdridiales, rather than stimulating Fe3+ respiring populations. Data were similar irrespective of the inoculum source, suggesting that Fe3+ and/or electron shuttling compounds select for rapid proliferation of fermentative genera when fermentable substrates are present, and increases the extent of xylose consumption and solvent production. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-13T06:28:16.22934-05:0
      DOI: 10.1002/bit.26494
  • Genome-wide identification of tolerance mechanisms towards p-coumaric acid
           in Pseudomonas putida
    • Authors: Patricia Calero; Sheila I. Jensen, Klara Bojanovič, Rebecca Lennen, Anna Koza, Alex T. Nielsen
      Abstract: The soil bacterium Pseudomonas putida KT2440 has gained increasing biotechnological interest due to its ability to tolerate different types of stress. Here, the tolerance of P. putida KT2440 towards eleven toxic chemical compounds was investigated. P. putida was found to be significantly more tolerant towards three of the eleven compounds when compared to Escherichia coli. Increased tolerance was for example found towards p-coumaric acid, an interesting precursor for polymerization with a significant industrial relevance. The tolerance mechanism was therefore investigated using the genome-wide approach, Tn-seq. Libraries containing a large number of miniTn5-Km transposon insertion mutants were grown in the presence and absence of p-coumaric acid, and the enrichment or depletion of mutants was quantified by high-throughput sequencing. Several genes, including the ABC transporter Ttg2ABC and the cytochrome c maturation system (ccm), were identified to play an important role in the tolerance towards p-coumaric acid of this bacterium. Most of the identified genes were involved in membrane stability, suggesting that tolerance towards p-coumaric acid is related to transport and membrane integrity. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-13T05:55:19.764119-05:
      DOI: 10.1002/bit.26495
  • Preferential capture of EpCAM-expressing extracellular vesicles on solid
           surfaces coated with an aptamer-conjugated zwitterionic polymer
    • Authors: Mitsutaka Yoshida; Kazuhiro Hibino, Satoshi Yamamoto, Sachiko Matsumura, Yasutomo Yajima, Kiyotaka Shiba
      Abstract: Extracellular vesicles (EVs) collectively represent small vesicles that are secreted from cells and carry biomolecules (e.g., miRNA, lncRNA, mRNA, proteins, lipids, metabolites, etc.) that originate in those cells. Body fluids, such as blood and saliva, include large numbers of EVs, making them potentially a rich source of diagnostic information. However, these EVs are mixtures of vesicles released from diseased tissues as well as from normal cells. This heterogeneous nature therefore blurs the clinical information obtainable from EV-based diagnosis. Here, we synthesized an EpCAM-affinity coating agent, which consists of a peptide aptamer for EpCAM and a zwitterionic MPC polymer, and have shown that this conjugate endowed the surfaces of inorganic materials with the preferential affinity to EpCAM-expressing EVs. This coating agent, designated as EpiVeta, could be useful as a coating for various diagnostic devices to allow concentration of cancer-related EVs from heterogeneous EV mixtures. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-06T08:01:06.735507-05:
      DOI: 10.1002/bit.26489
  • Engineering Escherichia coli for malate production by integrating modular
    • Authors: Cong Gao; Shihui Wang, Guipeng Hu, Liang Guo, Xiulai Chen, Peng Xu, Liming Liu
      Abstract: The application of rational design in reallocating metabolic flux to overproduce desired chemicals is always restricted by the native regulatory network. Here, we demonstrated that in vitro modular pathway optimization combined with in vivo multiplexed combinatorial engineering enables effective characterization of the bottleneck of a complex biosynthetic cascade and improves the output of the engineered pathway. As a proof of concept, we systematically identified the rate-limiting step of a five-gene malate biosynthetic pathway by combinatorially tuning the enzyme loads of a reconstituted biocatalytic reaction in a cell-free system. Using multiplexed CRISPR interference, we subsequently eliminated the metabolic constraints by rationally assigning an optimal gene expression pattern for each pathway module. The present engineered strain Escherichia coli B0013-47 exhibited a 2.3-fold increase in malate titer compared with that of the parental strain, with a yield of 0.85 mol/mol glucose in shake-flask culture and titer of 269 mM (36 g/L) in fed-batch cultivation. The strategy reported herein represents a powerful method for improving the efficiency of multi-gene pathways and advancing the success of metabolic engineering. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-04T08:25:33.720351-05:
      DOI: 10.1002/bit.26486
  • Metabolic profiles analysis of 1,3-propanediol production process by
           Clostridium butyricum through repeated batch fermentation coupled with
           activated carbon adsorption
    • Authors: Ai-Hui Zhang; Hao-Lin Liu, Shi-Yang Huang, You-Si Fu, Bai-Shan Fang
      Abstract: 1,3-propanediol production by Clostridium butyricum is a low productivity process due to the long time seed cultivation and thus hinders its industrial scale production. In the present study, repeated batch fermentation coupled with activated carbon adsorption strategy was first established which conduced not only to saving the time of seed cultivation and enhancing the productivity, but also to reducing the costs for the seed cultivation to achieve the purpose of 1,3-propanediol continuous production. The concentration of 1,3-propanediol from first to fourth cycle was 42.89, 45.78, 44.48, 42.39 (g/L), and the corresponding volumetric productivity was 2.14, 1.91, 1.85, 2.12 (g/L⋅h−1) respectively. More importantly, a relatively complete schematic diagram of the proposed metabolic pathways was firstly mapped out based on the intracellular metabolites analysis through GC-MS. At the same time, metabolic pathway and principal components analyses were carried out to give us deep insight into metabolic state. Many metabolites occurred to response to the stress in Cycle II. Even resting body formed and lipid accumulated owing to the worsening environment in the group without activated carbon in Cycle III. Thus, it demonstrated that activated carbon provided a favorable microenvironment for Clostridium butyricum in the repeated batch fermentation process to achieve the purpose of 1,3-propanediol continuous production. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-04T08:25:30.431677-05:
      DOI: 10.1002/bit.26488
    • Authors: Jiayi Sun; Le Zhao, Zengyi Shao, Jacqueline Shanks, Christie A. M. Peebles
      Abstract: The monoterpene indole alkaloids vindoline and catharanthine, which are exclusively synthesized in the medicinal plant Catharanthus roseus, are the two important precursors for the production of pharmaceutically important anti-cancer medicines vinblastine and vincristine. Hairy root culture is an ideal platform for alkaloids production due to its industrial scalability, genetic and chemical stability, and availability of genetic engineering tools. However, C. roseus hairy roots do not produce vindoline due to the lack of expression of the seven-step pathway from tabersonine to vindoline (Murata and De Luca, 2005). The present study describes the genetic engineering of the first two genes tabersonine 16-hydroxylase (T16H) and 16-O-methyl transferase (16OMT) in the missing vindoline pathway under the control of a glucocorticoid-inducible promoter to direct tabersonine toward vindoline biosynthesis in C. roseus hairy roots. In two transgenic hairy roots, the induced overexpression of T16H and 16OMT resulted in the accumulation of vindoline pathway metabolites 16-hydroxytabersonine and 16-methoxytabersonine. The levels of root-specific alkaloids, including lochnericine, 19-hydroxytabersonine and hörhammericine, significantly decreased in the induced hairy roots in comparison to the uninduced control lines. This suggests tabersonine was successfully channeled to the vindoline pathway away from the roots competing pathway based on the overexpression. Interestingly, another two new metabolites were detected in the induced hairy roots and proposed to be the epoxidized-16-hydroxytabersonine and lochnerinine. Thus the introduction of vindoline pathway genes in hairy roots can cause unexpected terpenoid indole alkaloids (TIA) profile alterations. Furthermore, we observed complex transcriptional changes in TIA genes and regulators detected by RT-qPCR which highlight the tight regulation of the TIA pathway in response to T16H and 16OMT engineering in C. roseus hairy roots. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-04T08:25:28.614035-05:
      DOI: 10.1002/bit.26487
  • Metabolic Phenotyping of CHO Cells Varying in Cellular Biomass
           Accumulation and Maintenance during Fed-Batch Culture
    • Authors: Alejandro Fernandez-Martell; Yusuf B. Johari, David C. James
      Abstract: CHO cell lines capable of high-level recombinant protein product biosynthesis during fed-batch culture are still generally obtained by intensive empirical screening of transfected cells rather than knowledge-guided cellular engineering. In this study, we investigate how CHO cell lines create and maintain cellular biosynthetic capacity during fed-batch culture to achieve the optimal combination of rapid exponential proliferation and extended maintenance of high cell biomass concentration. We perform a comparative meta-analysis of mitochondrial and glycolytic functions of 22 discrete parental CHO cell lineages varying in fed-batch culture performance to test the hypotheses that (i) “biomass-intensive” CHO cells exhibit conserved differences in metabolic programming and (ii) it is possible to isolate parental CHO cell lines with a biomass-intensive phenotype to support fed-batch bioproduction processes. We show that for most parental CHO cell lines, rapid proliferation and high late-stage culture performance are mutually exclusive objectives. However, quantitative dissection of mitochondrial and glycolytic functions revealed that a small proportion of clones utilize a conserved metabolic program that significantly enhances cellular glycolytic and mitochondrial oxidative capacity at the onset of late-stage culture. We reveal the central importance of dynamic metabolic re-programming to activate oxidative mitochondrial function as a necessary mechanism to support CHO cell biosynthetic performance during culture. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-28T07:55:36.248764-05:
      DOI: 10.1002/bit.26485
  • Identification of Key Residues Modulating the Stereoselectivity of Nitrile
           Hydratase towards rac-Mandelonitrile by Semi-rational Engineering
    • Authors: Zhongyi Cheng; Lukasz Peplowski, Wenjing Cui, Yuanyuan Xia, Zhongmei Liu, Jialei Zhang, Michihiko Kobayashi, Zhemin Zhou
      Abstract: Optically pure compounds are important in the synthesis of fine chemicals. Using directed evolution of enzymes to obtain biocatalysts that can selectively produce high-value chiral chemicals is often time-, money- and resource-intensive; traditional semi-rational designs based on structural data and docking experiments are still limited due to the lack of accurate selection of hot-spot residues. In this study, through ligand-protein collision counts based on steered molecular dynamics simulation, we accurately identified four residues related to improving nitrile hydratase stereoselectivity towards rac-mandelonitrile (MAN). All the four selected residues had numerous collisions with rac-MAN. Five mutants significantly shifting stereoselectivity towards (S)-MAN were obtained from site-saturation mutagenesis, one of them, at position βPhe37, exhibiting efficient production of (S)-MAN with 96.8% eep, was isolated and further analyzed. The increased pulling force observed during SMD simulation was found to be in good coincidence with the formation of hydrogen bonds between (R)-MAN and residue βHis37. (R)-MAN had to break these barriers to enter the active site of nitrile hydratase and S selectivity was thus improved. The results indicated that combining steered molecular dynamics simulation with a traditional semi-rational design significantly reduced the select range of hot-spot residues for the evolution of NHase stereoselectivity, which could serve as an alternative for the modulation of enzyme stereoselectivity. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-28T07:55:28.074477-05:
      DOI: 10.1002/bit.26484
  • Enzymatic Synthesis of Chiral Amino-Alcohols by Coupling Transketolase and
           Transaminase-Catalyzed Reactions in a Cascading Continuous-Flow
           Microreactor System
    • Authors: Pia Gruber; Filipe Carvalho, Marco P.C. Marques, Brian O'Sullivan, Fabiana Subrizi, Dragana Dobrijevic, John Ward, Helen C. Hailes, Pedro Fernandes, Roland Wohlgemuth, Frank Baganz, Nicolas Szita
      Abstract: Rapid biocatalytic process development and intensification continues to be challenging with currently available methods. Chiral amino-alcohols are of particular interest as they represent key industrial synthons for the production of complex molecules and optically pure pharmaceuticals. (2S,3R)-2-amino-1,3,4-butanetriol (ABT), a building block for the synthesis of protease inhibitors and detoxifying agents, can be synthesized from simple, non-chiral starting materials, by coupling a transketolase- and a transaminase-catalyzed reaction. However, until today, full conversion has not been shown and, typically, long reaction times are reported, making process modifications and improvement challenging. In this contribution, we present a novel microreactor-based approach based on free enzymes, and we report for the first time full conversion of ABT in a coupled enzyme cascade for both batch and continuous-flow systems. Using the compartmentalization of the reactions afforded by the microreactor cascade, we overcame inhibitory effects, increased the activity per unit volume, and optimized individual reaction conditions. The transketolase-catalyzed reaction was completed in under 10 minutes with a volumetric activity of 3.25 U mL−1. Following optimization of the transaminase-catalyzed reaction, a volumetric activity of 10.8 U mL−1 was attained which led to full conversion of the coupled reaction in 2 hours. The presented approach illustrates how continuous-flow microreactors can be applied for the design and optimization of biocatalytic processes. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-07T07:34:53.391206-05:
      DOI: 10.1002/bit.26470
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