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Journal Cover Biotechnology and Bioengineering
  [SJR: 1.633]   [H-I: 146]   [141 followers]  Follow
    
   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  [1589 journals]
  • 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
           applications
    • 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
           Strategies
    • 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
           oligosaccharyltransferases
    • 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
       
  • Cover Image, Volume 115, Number 1, January 2018
    • Authors: Catherine B. Matthews; Angel Kuo, Kerry R. Love, J. Christopher Love
      Abstract: Cover Legend: The cover image, by Catherine B. Matthews et al., is based on the Article Development of a general defined medium for Pichia pastoris,
      DOI 10.1002/bit.26440
      PubDate: 2017-11-22T09:34:26.468581-05:
       
  • 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
           mutagenesis
    • 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
       
  • Production of 1,2-propanediol in photoautotrophic Synechocystis is linked
           to glycogen turn-over
    • Authors: Christian David; Andreas Schmid, Lorenz Adrian, Annegret Wilde, Katja Bühler
      Abstract: We utilized a photoautotrophic organism to synthesize 1,2-propanediol from carbon dioxide and water fueled by light. A synthetic pathway comprising mgsA (methylglyoxal synthase), yqhD (aldehyde reductase), and adh (alcohol dehydrogenase) was inserted into Synechocystis sp. PCC6803 to convert dihydroxyacetone phosphate to methylglyoxal, which is subsequently reduced to acetol and then to 1,2-propanediol. 1,2-propanediol could be successfully produced by Synechocystis, at an approximate rate of 55 µmol h−1 gCDW−1. Surprisingly, maximal productivity was observed in the stationary phase. The production of 1,2-propanediol was clearly coupled to the turn-over of intracellular glycogen. Upon depletion of the glycogen pool, product formation stopped. Reducing the carbon flux to glycogen significantly decreased final product titers. Optimization of cultivation conditions allowed final product titers of almost 1 g L−1 (12 mM), which belongs to the highest values published so far for photoautotrophic production of this compound. This article is protected by copyright. All rights reserved
      PubDate: 2017-11-16T07:57:42.708615-05:
      DOI: 10.1002/bit.26468
       
  • 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
       
  • EXPRESSION OF TABERSONINE 16-HYDROXYLASE AND
           16-HYDROXYTABERSONINE-O-METHYLTRANSFERASE IN CATHARANTHUS ROSEUS HAIRY
           ROOTS
    • 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
       
  • Dynamic modelling of green algae cultivation in a photobioreactor for
           sustainable biodiesel production
    • Authors: Ehecatl Antonio del Rio-Chanona; Jiao Liu, Jonathan L. Wagner, Dongda Zhang, Yingying Meng, Song Xue, Nilay Shah
      Abstract: Biodiesel produced from microalgae has been extensively studied due to its potentially outstanding advantages over traditional transportation fuels. In order to facilitate its industrialisation and improve the process profitability, it is vital to construct highly accurate models capable of predicting the complex behaviour of the investigated biosystem for process optimisation and control, which forms the current research goal. Three original contributions are described in this paper. Firstly, a dynamic model is constructed to simulate the complicated effect of light intensity, nutrient supply and light attenuation on both biomass growth and biolipid production. Secondly, chlorophyll fluorescence, an instantly measurable variable and indicator of photosynthetic activity, is embedded into the model to monitor and update model accuracy especially for the purpose of future process optimal control, and its correlation between intracellular nitrogen content is quantified, which to the best of our knowledge has never been addressed so far. Thirdly, a thorough experimental verification is conducted under different scenarios including both continuous illumination and light/dark cycle conditions to testify the model predictive capability particularly for long-term operation, and it is concluded that the current model is characterised by a high level of predictive capability. Based on the model, the optimal light intensity for algal biomass growth and lipid synthesis is estimated. This work, therefore, paves the way to forward future process design and real-time optimisation. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-28T07:50:21.371759-05:
      DOI: 10.1002/bit.26483
       
  • A "Plug-n-play" Modular Metabolic System for the Production of
           Apocarotenoids
    • Authors: Congqiang Zhang; Xixian Chen, Nic D Lindley, Heng-Phon Too
      Abstract: Apocarotenoids, such as α-, β-ionone and retinol, have high commercial values in the food and cosmetic industries. The demand for natural ingredients has been increasing dramatically in recent years. However, attempts to overproduce β-ionone in microorganisms have been limited by the complexity of the biosynthetic pathway. Here, an Escherichia coli-based modular system was developed to produce various apocarotenoids. Incorporation of enzyme engineering approaches (N-terminal truncation and protein fusion) into modular metabolic engineering strategy significantly improved α-ionone production from 0.5 to 30 mg/L in flasks, producing 480 mg/L of α-ionone in fed-batch fermentation. By modifying apocarotenoid genetic module, this platform strain was successfully re-engineered to produce 32 mg/L and 500 mg/L of β-ionone in flask and bioreactor, respectively (> 80-fold higher than previously reported). Similarly, 33 mg/L of retinoids was produced in flask by reconstructing apocarotenoid module, demonstrating the versatility of the "plug-n-play" modular system. Collectively, this study highlights the importance of the strategy of simultaneous modular pathway optimization and enzyme engineering to overproduce valuable chemicals in microbes. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-27T09:00:32.720866-05:
      DOI: 10.1002/bit.26462
       
  • Effect of Culture Density on Biomass Production and Light Utilization
           Efficiency of Synechocystis sp. PCC 6803
    • Authors: Levi Straka; Bruce E. Rittmann
      Abstract: The viability of large-scale microalgae cultivation depends on providing optimal growth conditions, for which a key operational parameter is culture density. Using Synechocystis sp. PCC 6803, we conducted a series of fixed-density, steady-state experiments and one batch-growth experiment to investigate the role of culture density on biomass production and light utilization efficiency. In all cases, the fixed-density, steady-state experiments and batch-growth experiment showed good agreement. The highest biomass production rates (260 mg L−1 d−1) and efficiency for converting light energy to biomass (0.80 μg (μmol photons)−1) occurred together at a culture density near 760 mg L−1, which approximately corresponded to the lowest culture density where almost all incident light was absorbed. The ratio of OD680/OD735 increased with culture density up to the point of maximum productivity, where it plateaued (at a value of 2.4) for higher culture densities. This change in OD680/OD735 indicates a photoacclimation effect that depended on culture density. Very high culture densities led to a sharp decline in efficiency of biomass production per photons absorbed, likely due to a combination of increased decay relative to growth, metabolic changes due to cell-cell interactions, and photodamage due to mixing between regions with high light intensity and zero light intensity. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-24T08:26:21.687812-05:
      DOI: 10.1002/bit.26479
       
  • Comparative genomics and transcriptomics analysis-guided metabolic
           engineering of Propionibacterium acidipropionici for improved propionic
           acid production
    • Authors: Ningzi Guan; Bin Du, Jianghua Li, Hyun-dong Shin, Rachel R. Chen, Guocheng Du, Jian Chen, Long Liu
      Abstract: Acid stress induced by the accumulation of organic acids during the fermentation of propionibacteria is a severe limitation in the microbial production of propionic acid (PA). To enhance the acid resistance of strains, the tolerance mechanisms of cells must first be understood. In this study, comparative genomic and transcriptomic analyses were conducted on wild-type and acid-tolerant Propionibacterium acidipropionici to reveal the microbial response of cells to acid stress during fermentation. Combined with the results of previous proteomic and metabolomic studies, several potential acid-resistance mechanisms of P. acidipropionici were analyzed. Energy metabolism and transporter activity of cells were regulated to maintain pH homeostasis by balancing transmembrane transport of protons and ions; redundant protons were eliminated by enhancing the metabolism of certain amino acids for a relatively stable intracellular microenvironment; and protective mechanism of macromolecules were also induced to repair damage to proteins and DNA by acids. Transcriptomic data indicated that the synthesis of acetate and lactate were undesirable in the acid-resistant mutant, the expression of which was 2.21-fold downregulated. In addition, metabolomic data suggested that the accumulation of lactic acid and acetic acid reduced the carbon flow to PA and led to a decrease in pH. On this basis, we propose a metabolic engineering strategy to regulate the synthesis of lactic acid and acetic acid that will reduce by-products significantly and increase the PA yield by 12.2% to 10.31 ± 0.84 g/g DCW. Results of this study provide valuable guidance to understand the response of bacteria to acid stress and to construct microbial cell factories to produce organic acids by combining systems biology technologies with synthetic biology tools. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-24T08:26:18.511065-05:
      DOI: 10.1002/bit.26478
       
  • Directed evolution of polypropylene and polystyrene binding peptides
    • Authors: Kristin Rübsam; Lina Weber, Felix Jakob, Ulrich Schwaneberg
      Abstract: Surface functionalization of biological inert polymers (e.g. polypropylene PP; polystyrene PS) with material binding peptides facilitates an efficient immobilization of enzymes, bioactive peptides or antigens at ambient temperature in water. The developed robust directed evolution protocol enables to tailor polymer binding anchor peptides (PBPs) for efficient binding under application conditions. Key for a successful directed evolution campaign was to develop an epPCR protocol with a very high mutation frequency (60 mutations/kb) to ensure sufficient diversity in PBPs (47 aas LCI: "liquid chromatography peak I"; 44 aas TA2: "Tachystatin A2"). LCI and TA2 were genetically fused to the reporter egfp to quantify peptide binding on PP and PS by fluorescence analysis. The Peptide-Polymer evolution protocol (PePevo protocol) was validated in two directed evolution campaigns for two PBPs and polymers (LCI: PP; TA2: PS). Surfactants were used as selection pressure for improved PBP binders (non-ionic surfactant Triton X-100; 1 mM for LCI-PP // anionic surfactant LAS; 0.5 mM for TA2-PS). PePevo yielded an up to three fold improved PP-binder (LCI-M1-PP: I24T, Y29H, E42K and LCI-M2-PP: D31V, E42G) and an up to six fold stronger PS-binder (TA2-M1-PS: R3S, L6P, V12K, S15P, C29R, R30L, F33S, Y44H and TA2-M2-PS: F9C, C24S, G26D, S31G, C41S, Y44Q). This article is protected by copyright. All rights reserved
      PubDate: 2017-10-24T08:26:08.00098-05:0
      DOI: 10.1002/bit.26481
       
  • Effects of Scaffold Microstructure and Low Intensity Pulsed Ultrasound on
           Chondrogenic Differentiation of Human Mesenchymal Stem Cells
    • Authors: Mitra Aliabouzar; Se-jun Lee, Xuan Zhou, Grace Lijjie Zhang, Kausik Sarkar
      Abstract: The effects of low intensity pulsed ultrasound (LIPUS) on proliferation and chondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on 3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) scaffolds with varying pore geometries (square and hexagonal channels) were investigated. The scaffold with square pores resulted in higher hMSC growth and chondrogenic differentiation than a solid or a hexagonally porous scaffold. The optimal LIPUS parameters at 1.5 MHz were found to be 100 mW/cm2 and 20% duty cycle. LIPUS stimulation increased proliferation by up to 60% after 24 hours. For chondrogenesis, we evaluated key cartilage biomarkers abundant in cartilage tissue; glycosaminoglycan (GAG), type II collagen and total collagen. LIPUS stimulation enhanced GAG synthesis up to 16% and 11% for scaffolds with square and hexagonal patterns, respectively, after 2 weeks. Additionally, type II collagen production increased by 60% and 40% for the same patterns respectively under LIPUS stimulation after 3 weeks. These results suggest that LIPUS stimulation, which has already been approved by FDA for treatment of bone fracture, could be a highly efficient tool for tissue engineering in combination with 3D printing and hMSCs to regenerate damaged cartilage tissues. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-24T08:25:51.258604-05:
      DOI: 10.1002/bit.26480
       
  • Comparison of Purification Strategies for Antibodies Used in a Broad
           Spectrum Host Cell Protein Immunoassay
    • Authors: Phoebe A. Baldus; Matthew Brown, Richard S. Wright, Jessica A. Campbell, Scott Lute, Brittany Chavez, Kurt Brorson, Ned Mozier
      Abstract: Host cell proteins (HCPs) are a heterogeneous mixture of impurities that should be minimized in bulk preparations of biotechnologically produced medicines. Immunoassays are commonly used to detect and measure HCPs in therapeutic products, and a successful assay is directly dependent on the quality of the polyclonal antibodies (pAbs) used. These pAbs are enriched from antisera of animals immunized with a broad mixture of HCPs, but there is limited information regarding the best strategy for purification of these critical reagents. The use of protein A or protein G affinity chromatography results in purified pAbs that are not entirely HCP-specific, while the use of HCP affinity chromatography results in a more specific pAb population but may be harder to recover fully. In theory, both approaches have advantages and disadvantages for generating optimal reagents. In this study, we compared reagents from these two purification procedures using the same starting material, as well as those from a step-wise combination of the two by evaluating purity, concentration, reagent coverage by western blotting, and performance in an enzyme-linked immunosorbent assay (ELISA). This study demonstrates that pAbs purified by each of the methods are very similar in terms of sensitivity, the ability to recognize a broad range of HCPs, and overall performance in an ELISA measuring a range of HCPs in upstream process and final drug substance samples. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-24T08:25:35.088358-05:
      DOI: 10.1002/bit.26482
       
  • Enhanced pyruvate production in Candida glabrata by carrier engineering
    • Authors: Zhengshan Luo; Song Liu, Guocheng Du, Sha Xu, Jingwen Zhou, Jian Chen
      Abstract: Pyruvate is an important organic acid that plays a key role in the central metabolic pathway. Manipulating transporters is an efficient strategy to enhance production of target organic acids and a means to understand the effects of altered intracellular pyruvate content on global metabolic networks. Efforts have been made to manipulate mitochondrial pyruvate carrier (MPC) to transport pyruvate into different subcellular compartments in Candida glabrata to demonstrate the effects of the subcellular distribution of pyruvate on central carbon metabolism. By increasing the mitochondrial pyruvate content through enhancing the rate of pyruvate transport into mitochondria, a high central carbon metabolism rate, specific growth rate and specific pyruvate production rate were obtained. Comparing the intracellular pyruvate content of engineered and control strains showed that higher intracellular pyruvate levels were not conducive to improving pyruvate productivity or central carbon metabolism. Plasma membrane expression of MPCs significantly increased the expression levels of key rate-limiting glycolytic enzymes. Moreover, pyruvate production of CGΔura3-Sp-MPC1, CGΔura3-Sp-MPC2 and CGΔura3-Sp-MPC1-Sp-MPC2 increased 134.4%, 120.3% and 30.0%, respectively. In conclusion, lower intracellular pyruvate content enhanced central carbon metabolism and provided useful clues for improving the production of other organic acids in microorganisms. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-16T08:27:56.482169-05:
      DOI: 10.1002/bit.26477
       
  • Overproduction of MCL-PHA with high 3-Hydroxydecanoate Content
    • Authors: Jie Gao; Minh T. Vo, Juliana A. Ramsay, Bruce A. Ramsay
      Abstract: Methods of producing medium-chain-length poly-3-hydroxyalkanoate (mcl-PHA) with high content of the dominant subunit, 3-hydroxydecanoate (HD), were examined with an emphasis on a high yield of polymer from decanoic acid. High HD content was achieved by using a β-oxidation knockout mutant of Pseudomonas putida KT2440 (designated as P. putida DBA-F1) or by inhibiting β-oxidation with addition of acrylic acid to wild type P. putida KT2440 in carbon-limited, fed-batch fermentations. At a substrate feed ratio of decanoic acid and acetic acid to glucose (DAA:G) of 6:4 g/g, P. putida DBA-F1 accumulated significantly higher HD (97 mol%), but much lower biomass (8.5 g/L) and PHA (42% of dry biomass) than the wild type. Both biomass and PHA concentrations were improved by decreasing the ratio of DAA:G to 4:6. Moreover, when the substrate feed ratio was further decreased to 2:8, 18 g/L biomass containing 59% mcl-PHA consisting of 100 mol% HD was achieved. The yield of PHA from decanoic acid was 1.24 (g/g) indicating that de novo synthesis had contributed to production. Yeast extract and tryptone addition allowed the mutant strain to accumulate 74% mcl-PHA by weight with 97 mol% HD at a production rate of 0.41 g/L/h, at least twice that of published data for any β-oxidation knock-out mutant. Higher biomass concentration was achieved with acrylic acid inhibition of β-oxidation in the wild type but the HD content (84 mol%) was less than that of the mutant. A carbon balance showed a marked increase in supernantant organic carbon for the mutant indicating overflow metabolism. Increasing the dominant monomer content (HD) greatly increased melting point, crystallinity and rate of crystallization. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-14T08:45:41.966646-05:
      DOI: 10.1002/bit.26474
       
  • Metabolic reduction of resazurin; location within the cell for
           cytotoxicity assays
    • Authors: Jian Lin Chen; Terry W.J. Steele, David C. Stuckey
      Abstract: Resazurin is widely used as a metabolic indicator for living cells, however, there has been considerable debate in the literature with regards to the specific location in the cell where the non-fluorescent resazurin is reduced to the strongly fluorescent resorufin. This lack of clarity about the reduction site makes the use of resazurin reduction data in cytotoxicity studies difficult to interpret. In this study, E. faecalis, a Gram-positive and facultative anaerobic bacterial strain, and the most toxic chlorophenol, pentachlorophenol (PCP), were chosen as models for an anaerobe and toxicant, respectively. By studying the kinetics of resazurin reduction by E. faecalis after different treatments (cell disruption, bacterial filtration and pre-exposure to toxicant), we confirmed that resazurin reduction to resorufin by live Gram-positive and facultative anaerobic bacterial cells can only happen intracellularly under anaerobic conditions, while resorufin reduction to dihydroresorufin can happen both intracellularly and extracellularly. Based on the understanding of these fundamental mechanisms, we suggest that resazurin reduction can be used as a quick bioassay for measuring cytotoxicity. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-14T08:45:40.176993-05:
      DOI: 10.1002/bit.26475
       
  • Types of Cell Death and Apoptotic Stages in Chinese Hamster Ovary Cells
           Distinguished by Raman Spectroscopy
    • Authors: Shreyas Rangan; Sepehr Kamal, Stanislav O. Konorov, H. Georg Schulze, Michael W. Blades, Robin F. B. Turner, James M. Piret
      Abstract: Cell death is the ultimate cause of productivity loss in bioreactors that are used to produce therapeutic proteins. We investigated the ability of Raman spectroscopy to detect the onset and types of cell death for Chinese Hamster Ovary (CHO) cells - the most widely used cell type for therapeutic protein production. Raman spectroscopy was used to compare apoptotic, necrotic, autophagic and control CHO cells. Several specific nucleic acid-, protein- and lipid-associated marker bands within the 650-850 cm−1 spectral region were identified that distinguished among cells undergoing different modes of cell death; supporting evidence was provided by principal component analysis of the full spectral data. In addition to comparing the different modes of cell death, normal cells were compared to cells sorted at several stages of apoptosis, in order to explore the potential for early detection of apoptosis. Different stages of apoptosis could be distinguished via Raman spectroscopy, with multiple changes observed in nucleic acid peaks at early stages whereas an increase in lipid signals was a feature of late apoptosis/secondary necrosis. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-14T08:45:36.546999-05:
      DOI: 10.1002/bit.26476
       
  • A synthetic biology approach to transform Yarrowia lipolytica into a
           competitive biotechnological producer of β-carotene
    • Authors: Macarena Larroude; Ewelina Celinska, Alexandre Back, Stephan Thomas, Jean-Marc Nicaud, Rodrigo Ledesma-Amaro
      Abstract: The increasing market demands of β-carotene as colorant, antioxidant and vitamin precursor, requires novel biotechnological production platforms. Yarrowia lipolytica, is an industrial organism unable to naturally synthesize carotenoids but with the ability to produce high amounts of the precursor Acetyl-CoA. We first found that a lipid overproducer strain was capable of producing more β-carotene than a wild type after expressing the heterologous pathway. Thereafter, we developed a combinatorial synthetic biology approach base on Golden Gate DNA assembly to screen the optimum promoter-gene pairs for each transcriptional unit expressed. The best strain reached a production titer of 1.5 g/L and a maximum yield of 0.048 g/g of glucose in flask. β-carotene production was further increased in controlled conditions using a fed-batch fermentation. A total production of β-carotene of 6.5 g/L and 90 mg/g DCW with a concomitant production of 42.6 g/L of lipids was achieved. Such high titers suggest that engineered Y. lipolytica is a competitive producer organism of β-carotene. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-07T07:47:05.080132-05:
      DOI: 10.1002/bit.26473
       
  • Metabolic Engineering of Bacillus subtilis for Production of D-Lactic acid
    • Authors: Deepika Awasthi; Liang Wang. Mun Su Rhee, Qingzhao Wang, Diane Chauliac, Lonnie O. Ingram, K. T. Shanmugam
      Abstract: Poly lactic acid (PLA) based plastics is renewable, bio-based and biodegradable. Although present day PLA is composed of mainly L-LA, an L- and D- LA copolymer is expected to improve the quality of PLA and expand its use. To increase the number of thermotolerant microbial biocatalysts that produce D-LA, a derivative of Bacillus subtilis strain 168 that grows at 50°C was metabolically engineered. Since B. subtilis lacks a gene encoding D-lactate dehydrogenase (ldhA), five heterologous ldhA genes (B. coagulans ldhA and gldA101 and ldhA from three Lactobacillus delbrueckii) were evaluated. Corresponding D-LDHs were purified and biochemically characterized. Among these, D-LDH from L. delbrueckii subspecies bulgaricus supported the highest D-LA titer (about 1M) and productivity (2 g h−1 g cells−1) at 37°C (B. subtilis strain DA12). The D-LA titer at 48°C was about 0.6 M at a yield of 0.99 (g D-LA g−1glucose consumed). Strain DA12 also fermented glucose at 48°C in mineral salts medium to lactate at a yield of 0.89 g g−1 glucose and the D-lactate titer was 180 ± 4.5 mM. These results demonstrate the potential of B. subtilis as a platform organism for metabolic engineering for production of chemicals at 48°C that could minimize process cost. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-07T07:40:09.219152-05:
      DOI: 10.1002/bit.26472
       
  • Impact of carbon monoxide partial pressures on methanogenesis and medium
           chain fatty acids production during ethanol fermentation
    • Authors: Sofia Esquivel-Elizondo; Joseph Miceli, Cesar I. Torres, Rosa Krajmalnik-Brown
      Abstract: Medium-chain fatty acids (MCFA) are important biofuel precursors. Carbon monoxide (CO) is a sustainable electron and carbon donor for fatty acid elongation, since it is metabolized to MCFA precursors, it is toxic to most methanogens, and it is a waste product generated in the gasification of waste biomass. The main objective of this work was to determine if the inhibition of methanogenesis through the continuous addition of CO would lead to increased acetate or MCFA production during fermentation of ethanol. The effects of CO partial pressures (PCO; 0.08–0.3 atm) on methanogenesis, fatty acids production, and the associated microbial communities were studied in batch cultures fed with CO and ethanol. Methanogenesis was partially inhibited at PCO ≥ 0.11 atm. This inhibition lead to increased acetate production during the first phase of fermentation (0–19 days). However, a second addition of ethanol (day 19) triggered MCFA production only at PCO ≥ 0.11 atm, which probably occurred through the elongation of acetate with CO-derived ethanol and H2:CO2. Accordingly, during the second phase of fermentation (days 20-36), the distribution of electrons to acetate decreased at higher PCO, while electrons channeled to MCFA increased. Most probably, Acetobacterium, Clostridium, Pleomorphomonas, Oscillospira and Blautia metabolized CO to H2:CO2, ethanol and/or fatty acids, while Peptostreptococcaceae, Lachnospiraceae and other Clostridiales utilized these metabolites, along with the provided ethanol, for MCFA production. These results are important for biotechnological systems where fatty acids production are preferred over methanogenesis, such as in chain elongation systems and microbial fuel cells.Descriptive textContinuous addition of carbon monoxide (CO) (140–428 mmol/L) at CO partial pressures ≥ 0.11 atm to fermentation with small amounts of ethanol (22 mmol/L) inhibited methanogenesis and lead to elongation of acetate (produced from ethanol) to propionate, butyrate and medium-chain fatty acids (MCFA) with CO-derived H2:CO2 and ethanol. Clostridium, Peptostreptococcaceae, Lachnospiraceae, and other Clostridiales most likely partnered with carboxidotrophs, potentially Acetobacterium, Pleomorphomonas, Oscillospira and Blautia species, for valerate, caproate, and heptanoate production. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-07T07:35:00.330731-05:
      DOI: 10.1002/bit.26471
       
  • One-step affinity capture and precipitation for improved purification of
           an industrial monoclonal antibody using Z-ELP functionalized nanocages
    • Authors: Andrew Swartz; Xuankuo Xu, Steven Traylor, Zheng Jian Li, Wilfred Chen
      Abstract: Protein A chromatography has been identified as a potential bottleneck in the monoclonal antibody production platform, leading to increased interest in non-chromatographic capture technologies. Affinity precipitation using environmentally responsive, Z-domain-elastin-like polypeptide (Z-ELP) fusion proteins has been shown to be a promising alternative. However, elevated temperature and salt concentrations necessary for precipitation resulted in decreased antibody monomer content and reduced purification capacity. To improve upon the existing technology, we reported an enhanced affinity precipitation of antibodies by conjugating Z-ELP to a 25 nm diameter, self-assembled E2 protein nanocage (Z-ELP-E2). The enlarged scale of aggregate formation and IgG-triggered crosslinking through multi-valent binding significantly outperformed traditional Z-ELP-based methods. In the current work, we sought to develop an affinity precipitation process capable of purifying industrial monoclonal antibodies (mAbs) at ambient temperature with minimal added salt. We discovered that the mAb-nanocage complex aggregated within 10 minutes at room temperature without the addition of salt due to the enhanced multi-valent cross-linking. After precipitating out of solution, the complex remained insoluble under all wash buffers tested, and only resolubilized after a low pH elution. Through optimization of key process steps, the affinity precipitation yield and impurity clearance met or exceeded protein A chromatography performance with 95% yield, 3.7 logs host cell protein reduction, and>5 logs of DNA reduction from mAb cell culture. Because of the operational flexibility afforded by this one-step affinity capture and precipitation process, the Z-E2-ELP based approach has the potential to be a viable alternative to platform mAb purification. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-07T07:34:57.695597-05:
      DOI: 10.1002/bit.26467
       
  • 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
       
  • Biocatalytic conversion of cycloalkanes to lactones using an in-vivo
           cascade in Pseudomonas taiwanensis VLB120
    • Authors: Rohan Karande; Diego Salamanca, Andreas Schmid, Katja Buehler
      Abstract: Chemical synthesis of lactones from cycloalkanes is a multi-step process challenged by limitations in reaction efficiency (conversion and yield), atom economy (by-products) and environmental performance. A heterologous pathway comprising novel enzymes with compatible kinetics was designed in Pseudomonas taiwanensis VLB120 enabling in-vivo cascade for synthesizing lactones from cycloalkanes. The respective pathway included cytochrome P450 monooxygenase (CHX), cyclohexanol dehydrogenase (CDH), and cyclohexanone monooxygenase (CHXON) from Acidovorax sp. CHX100. Resting (non-growing) cells of the recombinant host P. taiwanensis VLB120 converted cyclohexane, cyclohexanol, and cyclohexanone to ϵ-caprolactone at 22 U gCDW−1, 80-100 U gCDW−1, and 170 U gCDW−1, respectively. Cyclohexane was completely converted with a selectivity of 65% for ϵ-caprolactone formation in 2h without accumulation of intermediate products. Promiscuity of the whole-cell biocatalyst gave access to analogous lactones from cyclooctane and cyclodecane. A total product concentration of 2.3 g L−1 and a total turnover number of 36720 was achieved over 5h with a biocatalyst concentration of 6.8 gCDW L−1. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-07T07:34:34.11286-05:0
      DOI: 10.1002/bit.26469
       
  • Comparison of Platform Host Cell Protein ELISA to Process-Specific Host
           Cell Protein ELISA
    • Authors: Feny Gunawan; Julie Nishihara, Peter Liu, Wendy Sandoval, Marty Vanderlaan, Heidi Zhang, Denise Krawitz
      Abstract: During expression of biotherapeutic proteins, complex mixtures of additional proteins are also produced by normal expression machinery of the host cell (termed “host cell proteins”, or HCP). HCPs pose a potential impact to patient safety and product efficacy, and therefore must be well-characterized and the ability of the process to clear these proteins must be demonstrated. Due to the complexity of HCP, the method(s) used for monitoring must be demonstrated to provide sufficient information about relevant proteins.The most commonly used analytical method for monitoring HCP is an enzyme-linked immunosorbent assay (ELISA). To ensure development of a suitable HCP ELISA, careful selection of critical reagents (anti-HCP antibodies and analytical standard) is crucial. During a recent major update to the manufacturing process of a biotherapeutic, we re-evaluated the suitability of the existing HCP ELISA for monitoring the HCP population in the updated process. In the evaluation, we compared a process-specific ELISA to a platform ELISA. Despite qualitative differences in the HCP profiles in 2D PAGE, LC-MS/MS showed that the HCP populations in the two analytical standards were similar. The process-specific HCP antibody had adequate HCP coverage, but was more sensitive to a few dominant proteins that were present in the upstream purification process. The platform HCP antibody had very broad coverage and additionally, could detect the majority of potential HCP impurities from this process. Furthermore, the platform HCP antibody was not biased toward a few dominant proteins and was more sensitive in the downstream purification process. Due to its broad HCP coverage and sensitivity, we conclude that our platform HCP ELISA method is superior to the process-specific HCP ELISA method. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-07T07:34:27.818299-05:
      DOI: 10.1002/bit.26466
       
  • A novel, smaller scaffold for Affitins: Showcase with binders specific for
           EpCAM
    • Authors: Valentina Kalichuk; Axelle Renodon-Cornière, Ghislaine Béhar, Federico Carrión, Gonzalo Obal, Mike Maillasson, Barbara Mouratou, Véronique Préat, Frédéric Pecorari
      Abstract: Affitins are highly stable engineered affinity proteins, originally derived from Sac7d and Sso7d, two 7 kDa DNA-binding polypeptides from Sulfolobus genera. Their efficiency as reagents for intracellular targeting, enzyme inhibition, affinity purification, immunolocalization and various other applications has been demonstrated. Recently, we have characterized the 7 kDa DNA-binding family, and Aho7c originating from Acidianus hospitalis was shown to be its smallest member with thermostability comparable to those of Sac7d and Sso7d. Here, after four rounds of selection by ribosome display against the human recombinant Epithelial Cell Adhesion Molecule (hrEpCAM), we obtained novel Aho7c-based Affitins. The binders were expressed in soluble form in E. coli, displayed high stability (up to 74°C; pH 0-12) and were shown to be specific for the hrEpCAM extracellular domain with picomolar affinities (KD = 110 pM). Thus, we propose Aho7c as a good candidate for the creation of Affitins with a 10% smaller size than the Sac7d-based ones (60 versus 66 amino acids). This article is protected by copyright. All rights reserved
      PubDate: 2017-10-04T10:01:03.964059-05:
      DOI: 10.1002/bit.26463
       
  • Effect of NADPH availability on free fatty acid production in E. coli
    • Authors: Wei Li; Hui Wu, Mai Li, Ka-Yiu San
      Abstract: Microbial conversion of renewable carbon sources to free fatty acids has attracted significant attention in recent years. Accumulation of free fatty acids in E. coli by overexpression of an acyl-ACP thioesterase which can break the fatty acid elongation has been well established. Various efforts have been made to increase fatty acid production in E. coli by enhancing the enzymes involved in the fatty acid synthesis cycle or host strain manipulations. The current study focused on the effect of NADPH availability on free fatty acids (FFAs) productivity. There are two reduction steps in the fatty acid elongation cycle which are catalyzed by beta keto-ACP reductase (FabG) and enoyl-ACP reductase (FabI), respectively. It is reported that FabI can use either NADH or NADPH as cofactor, while FabG only uses NADPH in E. coli. Fatty acid production dropped dramatically in the glucose-6-phosphate dehydrogenase (encoded by the zwf gene) deficient strain. Similarly, the pntB (which encodes one of the subunit of proton-translocating membrane bounded transhydrogenase PntAB) and udhA (which encodes the energy dependent cytoplasmic transhydrogenase UdhA) double mutant strain also showed an 88.8% decrease in free fatty acid production. Overexpression of PntAB and NadK restored the fatty acid production capability of these two mutant strains. These results indicated that the availability of NADPH played a very important role in fatty acid production. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-04T10:00:57.0558-05:00
      DOI: 10.1002/bit.26464
       
  • Enhancement of biomass and lipid productivity by overexpression of a bZIP
           transcription factor in Nannochloropsis salina
    • Authors: Sohee Kwon; Nam Kyu Kang, Hyun Gi Koh, Sung-Eun Shin, Bongsoo Lee, Byeong-ryool Jeong, Yong Keun Chang
      Abstract: Microalgae are considered as excellent platforms for biomaterial production that can replace conventional fossil fuel-based fuels and chemicals. Genetic engineering of microalgae is prerequisite to maximize production of materials and to reduce costs for the production. Transcription factors (TFs) are emerging as key regulators of metabolic pathways to enhance production of molecules for biofuels and other materials. TFs with the basic leucine zipper (bZIP) domain have been known as stress regulators and are associated with lipid metabolism in plants. We overexpressed a bZIP TF, NsbZIP1, in Nannochloropsis salina, and found that transformants showed enhanced growth with concomitant increase in lipid contents. The improved phenotypes were also notable under stress conditions including N limitation and high salt. To understand the mechanism underlying improved phenotypes, we analyzed expression patterns of predicted target genes involved in lipid metabolism via quantitative RT-PCR, confirming increases transcript levels. NsbZIP1 appeared to be one of type C bZIPs in plants that has been known to regulate lipid metabolism under stress. Taken together, we demonstrated that NsbZIP1 could improve both growth and lipid production, and TF engineering can serve as an excellent genetic engineering tool for production of biofuels and biomaterials in microalgae. This article is protected by copyright. All rights reserved
      PubDate: 2017-10-04T10:00:53.345198-05:
      DOI: 10.1002/bit.26465
       
  • Engineering of cell membrane to enhance heterologous production of
           hyaluronic acid in Bacillus subtilis
    • Authors: Adam W. Westbrook; Xiang Ren, Murray Moo-Young, C. Perry Chou
      Abstract: Hyaluronic acid (HA) is a high-value biopolymer used in the biomedical, pharmaceutical, cosmetic, and food industries. Current methods of HA production, including extraction from animal sources and streptococcal cultivations, are associated with high costs and health risks. Accordingly, the development of bioprocesses for HA production centered on robust 'Generally Recognized as Safe (GRAS)' organisms such as Bacillus subtilis is highly attractive. Here, we report the development of novel strains of B. subtilis in which the membrane cardiolipin (CL) content and distribution has been engineered to enhance the functional expression of heterologously expressed hyaluronan synthase of Streptococcus equisimilis (SeHAS), in turn, improving the culture performance for HA production. Elevation of membrane CL levels via overexpressing components involved in the CL biosynthesis pathway, and redistribution of CL along the lateral membrane via repression of the cell division initiator protein FtsZ resulted in increases to the HA titer of up to 204% and peak molecular weight of up to 2.2 MDa. Moreover, removal of phosphatidylethanolamine and neutral glycolipids from the membrane of HA-producing B. subtilis via inactivation of pssA and ugtP, respectively, has suggested the lipid dependence for functional expression of SeHAS. Our study demonstrates successful application of membrane engineering strategies to develop an effective platform for biomanufacturing of HA with B. subtilis strains expressing Class I streptococcal hyaluronan synthase. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-23T08:21:42.277218-05:
      DOI: 10.1002/bit.26459
       
  • Intracellular Response to Process Optimization and Impact on Productivity
           and Product Aggregates for a high-titer CHO Cell Process
    • Authors: Michael W. Handlogten; Allison Lee-O'Brien, Gargi Roy, Sophia V. Levitskaya, Raghavan Venkat, Shailendra Singh, Sanjeev Ahuja
      Abstract: A key goal in process development for antibodies is to increase productivity while maintaining or improving product quality. During process development of an antibody, titers were increased from 4 to 10 g/L while simultaneously decreasing aggregates. Process development involved optimization of media and feed formulations, feed strategy, and process parameters including pH and temperature. To better understand how CHO cells respond to process changes, the changes were implemented in a stepwise manner. The first change was an optimization of the feed formulation, the second was an optimization of the medium, and the third was an optimization of process parameters. Multiple process outputs were evaluated including cell growth, osmolality, lactate production, ammonium concentrations, antibody production, and aggregate levels. Additionally, detailed assessment of oxygen uptake, nutrient and amino acid consumption, extracellular and intracellular redox environment, oxidative stress, activation of the unfolded protein response (UPR) pathway, protein disulfide isomerase (PDI) expression and heavy and light chain mRNA expression provided in-depth understanding of the cellular response to the process changes. The results demonstrate that mRNA expression and UPR activation were unaffected by process changes, and that increased PDI expression and optimized nutrient supplementation are required for higher productivity processes. Furthermore, our findings demonstrate the role of extra- and intracellular redox environment on productivity and antibody aggregation. Processes using the optimized medium, with increased concentrations of redox modifying agents, had the highest overall specific productivity, reduced aggregate levels, and helped cells better withstand the high levels of oxidative stress associated with increased productivity. Specific productivities of different processes positively correlated to average intracellular values of total glutathione. Additionally, processes with the optimized media maintained an oxidizing intracellular environment that is important for correct disulfide bond pairing, which likely contributed to reduced aggregate formation. These findings shed important understanding into how cells respond to process changes and can be useful to guide future development efforts to enhance productivity and improve product quality. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-23T08:21:38.681208-05:
      DOI: 10.1002/bit.26460
       
  • Advancements in the design and scalable production of viral gene transfer
           vectors
    • Authors: David Sharon; Amine Kamen
      Abstract: The last 10 years have seen a rapid expansion in the use of viral gene transfer vectors, with approved therapies and late stage clinical trials underway for the treatment of genetic disorders, and multiple forms of cancer, as well as prevention of infectious diseases through vaccination. With this increased interest and widespread adoption of viral vectors by clinicians and biopharmaceutical industries, there is an imperative to engineer safer and more efficacious vectors, and develop robust, scalable and cost-effective production platforms for industrialization. This review will focus on major innovations in viral vector design and production systems for three of the most widely used viral vectors: Adenovirus, Adeno-Associated Virus, and Lentivirus. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-23T08:21:11.854017-05:
      DOI: 10.1002/bit.26461
       
  • Burkholderia cepacia lipase: A versatile catalyst in synthesis reactions
    • Authors: Daniel Alberto Sánchez; Gabriela Marta Tonetto, María Luján Ferreira
      Abstract: The lipase from Burkholderia cepacia, formerly known as Pseudomonas cepacia lipase, is a commercial enzyme in both soluble and immobilized forms widely recognized for its thermal resistance and tolerance to a large number of solvents and short-chain alcohols. The main applications of this lipase are in transesterification reactions and in the synthesis of drugs (because of the properties mentioned above). This review intends to show the features of this enzyme and some of the most relevant aspects of its use in different synthesis reactions. Also, different immobilization techniques together with the effect of various compounds on lipase activity are presented. This lipase shows important advantages over other lipases, especially in reaction media including solvents or reactions involving short-chain alcohols. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-23T08:15:24.987519-05:
      DOI: 10.1002/bit.26458
       
  • Reconstruction of Lignin and Hemicelluloses by Aqueous Ethanol
           Anti-solvents to Improve the Ionic Liquid-Acid Pretreatment Performance of
           Arundo donax Linn
    • Authors: Tingting You; Ruizhen Wang, Xueming Zhang, Shri Ramaswamy, Feng Xu
      Abstract: Ionic liquid (IL)-acid pretreatment is known to not only enhance the enzymatic hydrolysis efficiency of lignocellulose but also to generate deposits on the surface of fiber by conventional water regeneration, which retard the increment. In this study, ethanol aqueous solution regeneration was developed as a new method to change the substrates characteristics for IL-acid pretreatment and their effects on the enzymatic hydrolysis were evaluated. Following the IL-acid reaction, the biomass slurry was subjected to ethanol aqueous solution at various concentration. Results indicated that anti-solvent choice significantly influenced the reconstruction of both hemicelluloses and lignin as a result of the competition between water and ethanol. The partial removal of hemicelluloses and suitable lignin re-localization contributed to a more porous structure. Consequently, the cellulose digestibility of aqueous ethanol regenerated samples was dramatically enhanced to ∼100% and approximately 11-fold and 2-fold higher than that of untreated and conventional water regenerated pretreated samples, respectively. A giant leap in the initial rate of enzymatic hydrolysis was also detected in 50% ethanol aqueous solution regenerated samples and only about 10 h was needed to convert 80% of cellulose to glucose due to the appearance of cellulose II hydrate-like and more porous structure. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-23T08:15:22.544522-05:
      DOI: 10.1002/bit.26457
       
  • One pot synthesis of GDP-mannose by a multi-enzyme cascade for enzymatic
           assembly of lipid-linked oligosaccharides
    • Authors: Thomas F. T. Rexer; Anna Schildbach, Jan Klapproth, Angelika Schierhorn, Reza Mahour, Markus Pietzsch, Erdmann Rapp, Udo Reichl
      Abstract: Glycosylation of proteins is a key function of the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell-cell adhesion, blood-group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein-based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars. Here, we present a designed pathway consisting of five enzymes, glucokinase (Glk), phosphomannomutase (ManB), mannose-1-phosphate-guanyltransferase (ManC), inorganic pyrophosphatase (PmPpA) and 1-domain polyphosphate kinase 2 (1D-Ppk2) expressed in E. coli for the cell-free production and regeneration of GDP-mannose from mannose and polyphosphate with catalytic amounts of GDP and ADP. It was shown that GDP-mannose is produced at various conditions, i.e. pH 7–8, temperature 25–35°C and co-factor concentrations of 5–20 mM MgCl2. The maximum reaction rate of GDP-mannose achieved was 2.7 µM/min at 30°C and 10 mM MgCl2 producing 566 nmol GDP-mannose after a reaction time of 240 min. With respect to the initial GDP concentration (0.8 mM) this is equivalent to a yield of 71%. Additionally, the cascade was coupled to purified, transmembrane-deleted Alg1 (ALG1▵TM), the first mannosyltransferase in the ER-associated lipid-linked oligosaccharide (LLO) assembly. Thereby, in a one-pot reaction, phytanyl-PP-(GlcNAc)2-Man1 was produced with efficient nucleotide sugar regeneration for the first time. Phytanyl-PP-(GlcNAc)2-Man1 can serve as a substrate for the synthesis of LLO for the cell-free in vitro glycosylation of proteins. A high-performance anion exchange chromatography method with UV and conductivity detection (HPAEC-UV/CD) assay was optimized and validated to determine the enzyme kinetics. The established kinetic model enabled the optimization of the GDP-mannose regenerating cascade and can further be used to study coupling of the GDP-mannose cascade with glycosyltransferases. Overall, the study envisages a first step towards the development of a platform for the cell-free production of LLOs as precursors for in vitro glycoengineering of proteins.
      PubDate: 2017-09-18T07:00:25.321978-05:
      DOI: 10.1002/bit.26454
       
  • Improved performance of Pseudomonas putida in a bioelectrochemical system
           through overexpression of periplasmic glucose dehydrogenase
    • Authors: Shiqin Yu; Bin Lai, Manuel R. Plan, Mark P. Hodson, Endah A. Lestari, Hao Song, Jens O. Krömer
      Abstract: It was recently demonstrated that a bioelectrochemical system (BES) with a redox mediator allowed Pseudomonas putida to perform anoxic metabolism, converting sugar to sugar acids with high yield. However, the low productivity currently limits the application of this technology. To improve productivity the strain was optimized through improved expression of glucose dehydrogenase (GCD) and gluconate dehydrogenase (GAD). In addition, quantitative real-time RT-PCR analysis revealed the intrinsic self-regulation of GCD and GAD. Utilizing this self-regulation system, the single overexpression strain (GCD) gave an outstanding performance in the electron transfer rate and 2-ketogluconic acid (2KGA) productivity. The peak anodic current density, specific glucose uptake rate and 2KGA producing rate were 0.12 mA/cm2, 0.27 ± 0.02 mmol/gCDW/h and 0.25 ± 0.02 mmol/gCDW/h, which were 327%, 477% and 644% of the values of wild type P. putida KT2440, respectively. This work demonstrates that expression of periplasmic dehydrogenases involved in electron transfer can significantly improve productivity in the BES. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-18T02:55:21.33158-05:0
      DOI: 10.1002/bit.26433
       
  • Karyotype variation of CHO host cell lines over time in culture
           characterized by chromosome counting and chromosome painting
    • Authors: Sabine Vcelar; Vaibhav Jadhav, Michael Melcher, Norbert Auer, Astrid Hrdina, Rebecca Sagmeister, Kelley Heffner, Anja Puklowski, Michael Betenbaugh, Till Wenger, Friedrich Leisch, Martina Baumann, Nicole Borth
      Abstract: Genomic rearrangements are a common phenomenon in rapidly growing cell lines such as Chinese hamster ovary (CHO) cells, a feature that in the context of production of biologics may lead to cell line and product instability. Few methods exist to assess such genome wide instability. Here we use the population distribution of chromosome numbers per cell as well as chromosome painting to quantify the karyotypic variation in several CHO host cell lines.CHO-S, CHO-K1 8mM glutamine and CHO-K1 cells adapted to grow in media containing no glutamine were analyzed over up to 6 months in culture. All three cell lines were clearly distinguishable by their chromosome number distribution and by the specific chromosome rearrangements that were present in each population. Chromosome Painting revealed a predominant karyotype for each cell line at the start of the experiment, completed by a large number of variants present in each population. Over time in culture, the predominant karyotype changed for CHO-S and CHO-K1, with the diversity increasing and new variants appearing, while CHO-K1 0mM Gln preferred chromosome pattern increased in percent of the population over time. As control, Chinese hamster lung fibroblasts were shown to also contain an increasing number of variants over time in culture. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-16T07:55:39.319584-05:
      DOI: 10.1002/bit.26453
       
  • Development of a Formaldehyde Biosensor with Application to Synthetic
           Methylotrophy
    • Authors: Benjamin M. Woolston; Timothy Roth, Ishwar Kohale, David Liu, Gregory Stephanopoulos
      Abstract: Formaldehyde is a prevalent environmental toxin and a key intermediate in single carbon metabolism. The ability to monitor formaldehyde concentration is therefore of interest for both environmental monitoring and for metabolic engineering of native and synthetic methylotrophs, but current methods suffer from low sensitivity, complex workflows, or require expensive analytical equipment. Here we develop a formaldehyde biosensor based on the FrmR repressor protein and cognate promoter of E. coli. Optimization of the native repressor binding site and regulatory architecture enabled detection at levels as low as 1 µM. We then used the sensor to benchmark the in vivo activity of several NAD-dependent methanol dehydrogenase (Mdh) variants, the rate-limiting enzyme that catalyzes the first step of methanol assimilation. In order to use this biosensor to distinguish individuals in a mixed population of Mdh variants, we developed a strategy to prevent cross-talk by using glutathione as a formaldehyde sink to minimize intercellular formaldehyde diffusion. Finally, we apply this biosensor to balance expression of mdh and the formaldehyde assimilation enzymes hps and phi in an engineered E. coli strain to minimize formaldehyde build-up while also reducing the burden of heterologous expression. This biosensor offers a quick and simple method for sensitively detecting formaldehyde, and has the potential to be used as the basis for directed evolution of Mdh and dynamic formaldehyde control strategies for establishing synthetic methylotrophy. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-16T07:55:36.922457-05:
      DOI: 10.1002/bit.26455
       
  • Imaging stem cell distribution, growth, migration, and differentiation in
           3-D scaffolds for bone tissue engineering using mesoscopic fluorescence
           tomography
    • Authors: Qinggong Tang; Charlotte Piard, Jonathan Lin, Kai Nan, Ting Guo, John Caccamese, John Fisher, Yu Chen
      Abstract: Regenerative medicine has emerged as an important discipline that aims to repair injury or replace damaged tissues or organs by introducing living cells or functioning tissues. Successful regenerative medicine strategies will likely depend upon a simultaneous optimization strategy for the design of biomaterials, cell-seeding methods, cell-biomaterial interactions and molecular signaling within the engineered tissues. It remains a challenge to image three-dimensional (3-D) structures and functions of the cell-seeded scaffold in mesoscopic scale (>2∼3 mm). In this study, we utilized angled fluorescence laminar optical tomography (aFLOT), which allows depth-resolved molecular characterization of engineered tissues in 3-D to investigate cell viability, migration and bone mineralization within bone tissue engineering scaffolds in situ. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-16T07:55:33.642289-05:
      DOI: 10.1002/bit.26452
       
  • ΔFlucs: Brighter Photinus pyralis firefly luciferases identified by
           surveying consecutive single amino acid deletion mutations in a
           thermostable variant
    • Authors: Lisa M. Halliwell; Amit P. Jathoul, Jack P. Bate, Harley L. Worthy, James C. Anderson, Dafydd D. Jones, James A. H. Murray
      Abstract: The bright bioluminescence catalysed by Photinus pyralis firefly luciferase (Fluc) enables a vast array of life science research such as bioimaging in live animals and sensitive in vitro diagnostics. The effectiveness of such applications is improved using engineered enzymes that to date have been constructed using amino acid substitutions. We describe ΔFlucs: consecutive single amino acid deletion mutants within 6 loop structures of the bright and thermostable x11 Fluc. Deletion mutations are a promising avenue to explore new sequence and functional space and isolate novel mutant phenotypes. However, this method is often overlooked and to date there have been no surveys of the effects of consecutive single amino acid deletions in Fluc. We constructed a large semi-rational ΔFluc library and isolated significantly brighter enzymes after finding x11 Fluc activity was largely tolerant to deletions. Targeting an ‘omega-loop’ motif (T352-G360) significantly enhanced activity, altered kinetics, reduced Km for D-luciferin, altered emission colours and altered substrate specificity for redshifted analogue DL-infraluciferin. Experimental and in silico analyses suggested remodelling of the XXX-loop impacts on active site hydrophobicity to increase light yields. This work demonstrates the further potential of deletion mutations, which can generate useful Fluc mutants and broaden the palette of the biomedical and biotechnological bioluminescence enzyme toolbox. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-16T07:55:29.499696-05:
      DOI: 10.1002/bit.26451
       
  • Exploring cellular behaviour under transient gene expression and its
           impact on mAb productivity and Fc-glycosylation
    • Authors: Si Nga Sou; Ken Lee, Kalpana Nayyar, Karen M. Polizzi, Christopher Sellick, Cleo Kontoravdi
      Abstract: Transient gene expression (TGE) is a methodology employed in bioprocessing for the fast provision of recombinant protein material. Mild hypothermia is often introduced to overcome the low yield typically achieved with TGE and improve specific protein productivity. It is therefore of interest to examine the impact of mild hypothermic temperatures on both the yield and quality of transiently-expressed proteins and the relationship to changes in cellular processes and metabolism. In this study, we focus on the ability of a Chinese hamster ovary cell line to galactosylate a recombinant monoclonal antibody (mAb) product. Through experimentation and flux balance analysis, our results show that TGE in mild hypothermic conditions led to a 76% increase in qP compared to TGE at 36.5°C in our system. This increase is accompanied by increased consumption of nutrients and amino acids, together with increased production of intracellular nucleotide sugar species and higher rates of mAb galactosylation, despite a reduced rate of cell growth. The reduction in biomass accumulation allowed cells to redistribute their energy and resources towards mAb synthesis and Fc-glycosylation. Interestingly, the higher capacity of cells to galactosylate the recombinant product in TGE at 32°C appears not to have been assisted by the upregulation of galactosyltransferases (GalTs), but by the increased expression of N-acetylglucosaminyltransferase II (GnTII) in this cell line, which facilitated the production of bi-antennary glycan structures for further processing. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-16T07:55:24.875124-05:
      DOI: 10.1002/bit.26456
       
  • Recoding of synonymous genes to expand evolutionary landscapes requires
           control of secondary structure affecting translation
    • Authors: Jose Antonio Escudero; Aleksandra Nivina, Guillaume Cambray, Rocío López-Igual, Celine Loot, Didier Mazel
      Abstract: Synthetic DNA design needs to harness the many information layers embedded in a DNA string. We previously developed the Evolutionary Landscape Painter (ELP), an algorithm that exploits the degeneracy of the code to increase protein evolvability. Here, we have used ELP to recode the integron integrase gene (intI1) in two alternative alleles. Although synonymous, both alleles yielded less IntI1 protein and were less active in recombination assays than intI1. We spliced the three alleles and mapped the activity decrease to the beginning of alternative sequences. Mfold predicted the presence of more stable secondary structures in the alternative genes. Using synonymous mutations, we decreased their stability and recovered full activity. Following a design-build-test approach, we have now updated ELP to consider such structures and provide streamlined alternative sequences. Our results support the possibility of modulating gene activity through the ad hoc design of 5′ secondary structures in synthetic genes. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-12T06:30:43.311438-05:
      DOI: 10.1002/bit.26450
       
  • Systematic Optimization of L-Tryptophan Riboswitches for Efficient
           Monitoring of the Metabolite in Escherichia coli
    • Authors: Sungho Jang; Gyoo Yeol Jung
      Abstract: Riboswitches form a class of genetically-encoded sensor-regulators and are considered as promising tools for monitoring various metabolites. Functional parameters of a riboswitch, like dynamic or operational range, should be optimized before it is implemented in a specific application for monitoring the target molecule efficiently. However, optimization of a riboswitch was not straightforward and required detailed studies owing to its complex sequence-function relationship. Here, we present three approaches for tuning and optimization of functional parameters of a riboswitch using an artificial L-tryptophan riboswitch as an example. First, the constitutive expression level was adjusted to control the dynamic range of an L-tryptophan riboswitch. The dynamic range increased as the constitutive expression level increased. Then, the function of a riboswitch-encoded protein was utilized to connect the regulatory response of the riboswitch to another outcome for amplifying the dynamic range. Riboswitch-mediated control of the host cell growth enabled the amplification of the riboswitch response. Finally, L-tryptophan aptamers with different dissociation constants were employed to alter the operational range of the riboswitch. The dose-response curve was shifted towards higher L-tryptophan concentrations when an aptamer with higher dissociation constant was employed. All strategies were effective in modifying the distinct functional parameters of the L-tryptophan riboswitch, and they could be easily applied to optimization of other riboswitches owing to their simplicity. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-11T10:29:21.357807-05:
      DOI: 10.1002/bit.26448
       
  • Inhibition analysis of inhibitors derived from lignocellulose pretreatment
           on the metabolic activity of Zymomonas mobilis biofilm and planktonic
           cells and the proteomic responses
    • Authors: Tatsaporn Todhanakasem; Supanika Yodsanga, Apinya Sowatad, Pattanop Kanokratana, Pornthep Thanonkeo, Verawat Champreda
      Abstract: Lignocellulose pretreatment produces various toxic inhibitors that affect microbial growth, metabolism and fermentation. Zymomonas mobilis is an ethanologenic microbe that has been demonstrated to have potential to be used in lignocellulose biorefineries for bioethanol production. Z. mobilis biofilm has previously exhibited high potential to enhance ethanol production by presenting a higher viable cell number and higher metabolic activity than planktonic cells or free cells when exposed to lignocellulosic hydrolysate containing toxic inhibitors. However, there has not yet been a systematic study on the tolerance level of Z. mobilis biofilm compared to planktonic cells against model toxic inhibitors derived from lignocellulosic material. We took the first insight into the concentration of toxic compound (formic acid, acetic acid, furfural and 5-HMF) required to reduce the metabolic activity of Z. mobilis biofilm and planktonic cells by 25% (IC25), 50% (IC50), 75% (IC75) and 100% (IC100). Z. mobilis strains ZM4 and TISTR 551 biofilm were two- to three fold more resistant to model toxic inhibitors than planktonic cells. Synergetic effects were found in the presence of formic acid, acetic acid, furfural and 5-HMF. The IC25 of Z. mobilis ZM4 biofilm and TISTR 551 biofilm were 57 mM formic acid, 155 mM acetic acid, 37.5 mM furfural and 6.4 mM 5-HMF, and 225 mM formic acid, 291 mM acetic acid, 51 mM furfural and 41 mM 5-HMF, respectively. There was no significant difference found between proteomic analysis of the stress response to toxic inhibitors of Z. mobilis biofilm and planktonic cells on ZM4. However, TISTR 551 biofilms exhibited two proteins (molecular chaperone DnaK and 50S ribosomal protein L2) that were up-regulated in the presence of toxic inhibitors. TISTR 551 planktonic cells possessed two types of protein in the group of 30S ribosomal proteins and motility proteins that were up-regulated. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-11T10:29:19.216731-05:
      DOI: 10.1002/bit.26449
       
  • Fabricating PLGA microparticles with high loads of the small molecule
           antioxidant N-acetylcysteine that rescue oligodendrocyte progenitor cells
           from oxidative stress
    • Authors: Nicholas P. Murphy; Kyle J. Lampe
      Abstract: Reactive oxygen species (ROS), encompassing all oxygen radical or non-radical oxidizing agents, play key roles in disease progression. Controlled delivery of antioxidants is therapeutically relevant in such oxidant-stressed environments. Encapsulating small hydrophilic molecules into hydrophobic polymer microparticles via traditional emulsion methods has long been a challenge due to rapid mass transport of small molecules out of particle pores. We have developed a simple alteration to the existing water-in-oil-in-water (W/O/W) drug encapsulation method that dramatically improves loading efficiency: doping external water phases with drug to mitigate drug diffusion out of the particle during fabrication. 0.6 to 0.9 µm diameter PLGA microparticles were fabricated, encapsulating high loads of the antioxidant N-acetylcysteine (NAC), and released active, ROS-scavenging NAC for up to five weeks. Encapsulation efficiencies, normalized to the theoretical load of traditional encapsulation without doping, ranged from 96 to 400%, indicating that NAC-loaded external water phases not only prevented drug loss due to diffusion, but also doped the particles with additional drug. Antioxidant-doped particles positively affected the metabolism of oligodendrocyte progenitor cells (OPCs) under H2O2-mediated oxidative stress when administered both before (protection) or after (rescue) injury. Antioxidant doped particles improved outcomes of OPCs experiencing multiple doses of H2O2 by increasing the intracellular glutathione content and preserving cellular viability relative to the injury control. Furthermore, antioxidant-doped particles preserve cell number, number of process extensions, cytoskeletal morphology, and nuclear size of H2O2-stressed OPCs relative to the injury control. These NAC-doped particles have the potential to provide temporally-controlled antioxidant therapy in neurodegenerative disorders such as multiple sclerosis that are characterized by continuous oxidative stress. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-05T04:06:40.140696-05:
      DOI: 10.1002/bit.26443
       
  • Constructing arabinofuranosidases for dual arabinoxylan debranching
           activity
    • Authors: Weijun Wang; Nikola Andric, Cody Sarch, Bruno Teixeira Silva, Maija Tenkanen, Emma R. Master
      Abstract: Enzymatic conversion of arabinoxylan requires α-L-arabinofuranosidases able to remove α-L-arabinofuranosyl residues (α-L-Araf) from both mono- and double-substituted D-xylopyranosyl residues (Xylp) in xylan (i.e., AXH-m and AXH-d activity). Herein, SthAbf62A (a family GH62 α-L-arabinofuranosidase with AXH-m activity) and BadAbf43A (a family GH43 α-L-arabinofuranosidase with AXH-d3 activity), were fused to create SthAbf62A-BadAbf43A and BadAbf43A-SthAbf62A. Both fusion enzymes displayed dual AXH-m,d and synergistic activity towards native, highly branched wheat arabinoxylan (WAX). When using a customized arabinoxylan substrate comprising mainly α-(13)-L-Araf and α-(12)-L-Araf substituents attached to disubstituted Xylp (d-2,3-WAX), the specific activity of the fusion enzymes was twice that of enzymes added as separate proteins. Moreover, the SthAbf62A-BadAbf43A fusion removed 83% of all α-L-Araf from WAX after a 20 h treatment. 1H NMR analyses further revealed differences in SthAbf62A-BadAbf43 rate of removal of specific α-L-Araf substituents from WAX, where 9.4 times higher activity was observed towards d-α-(13)-L-Araf compared to m-α-(13)- L-Araf positions. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-04T02:10:41.567801-05:
      DOI: 10.1002/bit.26445
       
  • Power Input Effects on Degeneration in Prolonged Penicillin Chemostat
           Cultures: A Systems Analysis at Flux, Residual Glucose, Metabolite and
           Transcript Levels
    • Authors: Guan Wang; Baofeng Wu, Junfei Zhao, Cees Haringa, Jianye Xia, Ju Chu, Yingping Zhuang, Siliang Zhang, Joseph J. Heijnen, Walter van Gulik, Amit T. Deshmukh, Henk J. Noorman
      Abstract: In the present work, by performing chemostat experiments at 400 and 600 RPM, two typical power inputs representative of industrial penicillin fermentation (P/V, 1.00 kW/m3 in more remote zones and 3.83 kW/m3 in the vicinity of the impellers, respectively) were scaled-down to bench-scale bioreactors. It was found that at 400 RPM applied in prolonged glucose-limited chemostat cultures, the previously reported degeneration of penicillin production using an industrial Penicillium chrysogenum strain was virtually absent. To investigate this, the cellular response was studied at flux (stoichiometry), residual glucose, intracellular metabolite and transcript levels. At 600 RPM, 20% more cell lysis was observed and the increased degeneration of penicillin production was accompanied by a 22% larger ATP gap and an unexpected 20-fold decrease in the residual glucose concentration (Cs, out). At the same time, the biomass specific glucose consumption rate (qs) did not change but the intracellular glucose concentration was about 6-fold higher, which indicates a change to a higher affinity glucose transporter at 600 RPM. In addition, power input differences cause differences in the diffusion rates of glucose and the calculated Batchelor diffusion length scale suggests the presence of a glucose diffusion layer at the glucose transporting parts of the hyphae, which was further substantiated by a simple proposed glucose diffusion-uptake model. By analysis of calculated mass action ratios (MARs) and energy consumption, it indicated that at 600 RPM glucose sensing and signal transduction in response to the low Cs, out appear to trigger a gluconeogenic type of metabolic flux rearrangement, a futile cycle through the pentose phosphate pathway (PPP) and a declining redox state of the cytosol. In support of the change in glucose transport and degeneration of penicillin production at 600 RPM, the transcript levels of the putative high-affinity glucose/hexose transporter genes Pc12g02880 and Pc06g01340 increased 3.5 and 3.3-fold, respectively, and those of the pcbC gene encoding isopenicillin N-synthetase (IPNS) were more than 2-fold lower in the time range of 100 to 200 h of the chemostat cultures. Summarizing, changes at power input have unexpected effects on degeneration and glucose transport, and result in significant metabolic rearrangements. These findings are relevant for the industrial production of penicillin, and other fermentations with filamentous microorganisms. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-02T05:35:22.556125-05:
      DOI: 10.1002/bit.26447
       
  • Development of a general defined medium for Pichia pastoris
    • Authors: Catherine Bartlett Matthews; Angel Kuo, Kerry Routenberg Love, J. Christopher Love
      Abstract: Pichia pastoris is widely used as a host for recombinant protein production. More than 500 proteins have been expressed in the organism at a variety of cultivation scales, from small shake flasks to large bioreactors. Large-scale fermentation strategies typically employ chemically-defined growth medium because of its greater batch-to-batch consistency and in many cases, lower costs compared to complex medium. For biopharmaceuticals, defined growth medium may also simplify downstream purification and regulatory documentation. Standard formulations of defined media for Pichia pastoris are minimal ones that lack the metabolic intermediates provided by complex components such as peptone and yeast extract. As a result, growth rates and per-cell productivities are significantly lower than in complex medium. We have designed a rich defined medium (RDM) for Pichia pastoris by systematically evaluating nutrients of increasing complexity and identifying those that are most critical for growth. We have also demonstrated that using RDM for expression of three heterologous proteins yields titers comparable to, or higher than, those in standard complex medium. Rich defined medium improves productivity of Pichia pastoris fermentations and its development demonstrates the usefulness of transcriptomics to accelerate process development for new molecules. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-02T05:30:37.849949-05:
      DOI: 10.1002/bit.26440
       
  • Countercurrent Staged Diafiltration for Formulation of High Value Proteins
    • Authors: Anirudh Nambiar; Ying Li, Andrew L. Zydney
      Abstract: A number of groups have studied the application of continuous bioreactors and continuous chromatographic systems as part of efforts to develop an integrated continuous biomanufacturing process. The objective of this study was to examine the feasibility of using a countercurrent staged diafiltration process for continuous protein formulation with reduced buffer requirements. Experiments were performed using a polyclonal immunoglobulin (IgG) with CadenceTM Inline Concentrators. Model equations were developed for the product yield, impurity removal, and buffer requirements as a function of the number of stages and the stage conversion (ratio of permeate to feed flow rate). Data from a countercurrent two-stage system were in excellent agreement with model calculations, demonstrating the potential of using countercurrent staged diafiltration for protein formulation. Model simulations demonstrated the importance of the countercurrent staging on both the extent of buffer exchange and the amount of buffer required per kg of formulated product. The staged diafiltration process not only provides for continuous buffer exchange, it could also provide significant reductions in the number of pump passes while providing opportunities for reduced buffer requirements. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-02T05:30:35.586239-05:
      DOI: 10.1002/bit.26441
       
  • Glucose-Stimulated Insulin Release: Parallel Perifusion Studies of Free
           and Hydrogel Encapsulated Human Pancreatic Islets
    • Authors: Peter Buchwald; Alejandro Tamayo-Garcia, Vita Manzoli, Alice A. Tomei, Cherie L. Stabler
      Abstract: To explore the effects immune-isolating encapsulation has on the insulin secretion of pancreatic islets and to improve our ability to quantitatively describe the glucose-stimulated insulin release (GSIR) of pancreatic islets, we conducted dynamic perifusion experiments with isolated human islets. Free (unencapsulated) and hydrogel encapsulated islets were perifused, in parallel, using an automated multi-channel system that allows sample collection with high temporal resolution. Results indicated that free human islets secrete less insulin per unit mass or islet equivalent (IEQ) than murine islets and with a less pronounced first-phase peak. While small microcapsules (d ≈ 700 µm) caused only a slightly delayed and blunted first-phase insulin response compared to unencapsulated islets, larger capsules (d ≈ 1800 µm) completely blunted the first-phase peak and decreased the total amount of insulin released. Experimentally obtained insulin time-profiles were fitted with our complex insulin secretion computational model. This allowed further fine-tuning of the hormone-release parameters of this model, which was implemented in COMSOL Multiphysics to couple hormone secretion and nutrient consumption kinetics with diffusive and convective transport. The results of these GSIR experiments, which were also supported by computational modeling, indicate that larger capsules unavoidably lead to dampening of the first-phase insulin response and to a sustained-release type insulin secretion that can only slowly respond to changes in glucose concentration. Bioartificial pancreas type devices can provide long-term and physiologically desirable solutions only if immunoisolation and biocompatibility considerations are integrated with optimized nutrient diffusion and insulin release characteristics by design. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-02T05:30:33.336225-05:
      DOI: 10.1002/bit.26442
       
  • Temperature-dependent dynamic control of the TCA cycle increases
           volumetric productivity of itaconic acid production by Escherichia coli
    • Authors: Björn-Johannes Harder; Katja Bettenbrock, Steffen Klamt
      Abstract: Based on the recently constructed E. coli itaconic acid production strain ita23, we aimed to improve the productivity by applying a two-stage process strategy with decoupled production of biomass and itaconic acid. We constructed a strain ita32 (MG1655 ▵aceA ▵pta ▵pykF ▵pykA pCadCs), which, in contrast to ita23, has an active tricarboxylic acid (TCA) cycle and a fast growth rate of 0.52 h−1 at 37°C, thus representing an ideal phenotype for the first stage, the growth phase. Subsequently we implemented a synthetic genetic control allowing the downregulation of the TCA cycle and thus the switch from growth to itaconic acid production in the second stage. The promoter of the isocitrate dehydrogenase was replaced by the Lambda promoter (pR) and its expression was controlled by the temperature-sensitive repres-sor CI857 which is active at lower temperatures (30°C). With glucose as substrate, the respective strain ita36A grew with a fast growth rate at 37°C and switched to production of itaconic acid at 28°C. To study the impact of the process strategy on productivity we performed one-stage and two-stage bioreactor cultivations. The two-stage process enabled fast formation of biomass resulting in improved peak productivity of 0.86 g/L/h (+48%) and volumetric productivity of 0.39 g/L/h (+22%) in comparison to the one-stage process. With our dynamic production strain, we also resolved the glutamate auxotrophy of ita23 and increased the itaconic acid titer to 47 g/L.The temperature-dependent activation of gene expression by the Lambda promoters (pR/pL) has been frequently used to improve protein or, in a few cases, metabolite production in two-stage processes. Here we demonstrate that the system can be as well used in the opposite direction to selectively knock-down an essential gene (icd) in E. coli to design a two-stage process for improved volumetric productivity. The control by temperature avoids expensive inducers and has the potential to be generally used to improve cell factory performance. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-02T05:30:28.594414-05:
      DOI: 10.1002/bit.26446
       
  • Dry biorefining maximizes the potentials of simultaneous saccharification
           and co-fermentation for cellulosic ethanol production
    • Authors: Gang Liu; Qiang Zhang, Hongxing Li, Abdul Sattar Qureshi, Jian Zhang, Xiaoming Bao, Jie Bao
      Abstract: Despite the well-recognized merits of simultaneous saccharification and co-fermentation (SSCF) on relieving sugar product inhibition on cellulase activity, a practical concomitance difficulty of xylose with inhibitors in the pretreated lignocellulose feedstock prohibits the essential application of SSCF for cellulosic ethanol fermentation. To maximize the SSCF potentials for cellulosic ethanol production, a dry biorefining approach was proposed starting from dry acid pretreatment, disk milling and biodetoxification of lignocellulose feedstock. The successful SSCF of the inhibitor free and xylose conserved lignocellulose feedstock after dry biorefining reached a record high ethanol titer at moderate cellulase usage and minimum wastewater generation. For wheat straw, 101.4 g/L of ethanol (equivalent to 12.8% in volumetric percentage) was produced with the overall yield of 74.8% from cellulose and xylose, in which the xylose conversion was 73.9%, at the moderate cellulase usage of 15 mg protein per gram cellulose. For corn stover, 85.1 g/L of ethanol (equivalent to 10.8% in volumetric percentage) is produced with the overall conversion of 84.7% from cellulose and xylose, in which the xylose conversion was 87.7%, at the minimum cellulase usage of 10 mg protein per gram cellulose. Most significantly, the SSCF operation achieved the high conversion efficiency by generating the minimum amount of wastewater. Both the fermentation efficiency and the wastewater generation in the current dry biorefining for cellulosic ethanol production are very close to that of corn ethanol production, indicating that the technical gap between cellulosic ethanol and corn ethanol has been gradually filled by the advancing biorefining technology. This article is protected by copyright. All rights reserved
      PubDate: 2017-09-02T05:30:25.978223-05:
      DOI: 10.1002/bit.26444
       
  • Cell-Free Production of a Therapeutic Protein: Expression, Purification,
           and Characterization of Recombinant Streptokinase Using a CHO Lysate
    • Authors: Kevin Tran; Chandrasekhar Gurramkonda, Merideth A Cooper, Manohar Pilli, Joseph Tarris, Nick Selock, Tzu-Chiang Han, Michael Tolosa, Adil Zuber, Chariz Peñalber-Johnstone, Christina Dinkins, Niloufar Pezeshk, Yordan Kostov, Douglas D. Frey, Leah Tolosa, David Wood, Govind Rao
      Abstract: The use of cell-free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell-free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFPi and streptokinase in a cell-free system using instrumented mini-bioreactors for highly reproducible protein production. We achieved recombinant protein production (∼600 µg/mL of tGFP and 500 µg/mL streptokinase in 2.5 h of expression time, comparable to previously reported yields for cell-free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare these to a tag-free self-cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose-level production of therapeutic proteins at the point-of-care. This article is protected by copyright. All rights reserved
      PubDate: 2017-08-26T06:05:21.957277-05:
      DOI: 10.1002/bit.26439
       
  • A metabolic engineering strategy for producing free fatty acids by the
           Yarrowia lipolytica yeast based on impairment of glycerol metabolism
    • Authors: Evgeniya Y. Yuzbasheva; Elizaveta B. Mostova, Natalia I. Andreeva, Tigran V. Yuzbashev, Alexander S. Fedorov, Irina A. Konova, Sergey P. Sineoky
      Abstract: In recent years, bio-based production of free fatty acids from renewable resources has attracted attention for their potential as precursors for the production of biofuels and biochemicals. In this study, the oleaginous yeast Yarrowia lipolytica was engineered to produce free fatty acids by eliminating glycerol metabolism. Free fatty acid production was monitored under lipogenic conditions with glycerol as a limiting factor. Firstly, the strain W29 (Δgpd1), which is deficient in glycerol synthesis, was obtained. However, W29 (Δgpd1) showed decreased biomass accumulation and glucose consumption in lipogenic medium containing a limiting supply of glycerol. Analysis of substrate utilization from a mixture of glucose and glycerol by the parental strain W29 revealed that glycerol was metabolized first and glucose utilization was suppressed. Thus, the Δgpd1Δgut2 double mutant, which is deficient also in glycerol catabolism, was constructed. In this genetic background, growth was repressed by glycerol. Oleate toxicity was observed in the Δgpd1Δgut2Δpex10 triple mutant strain which is deficient additionally in peroxisome biogenesis. Consequently, two consecutive rounds of selection of spontaneous mutants were performed. A mutant released from growth repression by glycerol was able to produce 136.8 mg L−1 of free fatty acids in a test tube, whereas the wild type accumulated only 30.2 mg L−1. Next, an isolated oleate–resistant strain produced 382.8 mg L−1 of free fatty acids. Finely, acyl-CoA carboxylase gene (ACC1) over-expression resulted to production of 1436.7 mg L−1 of free fatty acids. The addition of dodecane promoted free fatty acid secretion and enhanced the level of free fatty acids up to 2033.8 mg L−1 during test tube cultivation. This article is protected by copyright. All rights reserved
      PubDate: 2017-08-19T06:25:19.212656-05:
      DOI: 10.1002/bit.26402
       
  • Incorporating LsrK AI-2 Quorum Quenching Capability in a Functionalized
           Biopolymer Capsule
    • Authors: Melissa K Rhoads; Pricila Hauk, Jessica Terrell, Chen-Yu Tsao, Hyuntaek Oh, Srinivasa R. Raghavan, Sheref S. Mansy, Gregory F Payne, William E Bentley
      Abstract: Antibacterial resistance is an issue of increasing severity as current antibiotics are losing their effectiveness and fewer antibiotics are being developed. New methods for combating bacterial virulence are required. Modulating molecular communication among bacteria can alter phenotype, including attachment to epithelia, biofilm formation and even toxin production. Intercepting and modulating communication networks provide a means to attenuate virulence without directly interacting with the bacteria of interest. In this work, we target communication mediated by the quorum sensing (QS) bacterial autoinducer-2, AI-2. We have assembled a capsule of biological polymers alginate and chitosan, attached an AI-2 processing kinase, LsrK, and provided substrate, ATP, for enzymatic alteration of AI-2 in culture fluids. Correspondingly, AI-2 mediated QS activity is diminished. All components of this system are “biofabricated” – they are biologically derived and their assembly is accomplished using biological means. Initially, component quantities and kinetics were tested as assembled in microtiter plates. Subsequently, the identical components and assembly means were used to create the “artificial cell” capsules. The functionalized capsules, when introduced into populations of bacteria, alter the dynamics of the AI-2 bacterial communication, attenuating QS activated phenotypes. We envision the assembly of these and other capsules or similar materials, as means to alter QS activity in a biologically compatible manner and in many environments, including in humans. This article is protected by copyright. All rights reserved
      PubDate: 2017-08-07T04:30:35.310416-05:
      DOI: 10.1002/bit.26397
       
  • Overproduction of L-tryptophan via simultaneous feed of glucose and
           anthranilic acid from recombinant E.coli W3110: kinetic modelling and
           process scale-up
    • Authors: Keju Jing; Yuanwei Tang, Chuanyi Yao, Ehecatl Antonio del Rio-Chanona, Xueping Ling, Dongda Zhang
      Abstract: L-tryptophan is an essential amino acid widely used in food and pharmaceutical industries. However, its production via Escherichia coli fermentation suffers severely from both low glucose conversion efficiency and acetic acid inhibition, and to date effective process control methods have rarely been explored to facilitate its industrial scale production. To resolve these challenges, in the current research an engineered strain of Escherichia coli was used to overproduce L-tryptophan. To achieve this, a novel dynamic control strategy which incorporates an optimised anthranilic acid feeding into a dissolved oxygen-stat (DO-stat) glucose feeding framework was proposed for the first time. Three original contributions were observed. Firstly, compared to previous DO control methods, the current strategy was able to inhibit completely the production of acetic acid, and its glucose to L-tryptophan yield reached 0.211 g/g, 62.3% higher than any previously reported. Secondly, a rigorous kinetic model was constructed to simulate the underlying biochemical process and identify the effect of anthranilic acid on both glucose conversion and L-tryptophan synthesis. Finally, a thorough investigation was conducted to testify the capability of both the kinetic model and the novel control strategy for process scale-up. It was found that the model possesses great predictive power, and the presented strategy achieved the highest glucose to L-tryptophan yield (0.224 g/g) ever reported in large scale processes, which approaches the theoretical maximum yield of 0.227 g/g. This research, therefore, paves the way to significantly enhance the profitability of the investigated bioprocess. This article is protected by copyright. All rights reserved
      PubDate: 2017-08-07T04:30:20.569189-05:
      DOI: 10.1002/bit.26398
       
  • Biotechnology and Bioengineering: Volume 115, Number 1, January 2018
    • Pages: 1 - 5
      PubDate: 2017-11-22T09:34:27.433223-05:
      DOI: 10.1002/bit.26410
       
 
 
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