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Journal Cover Biotechnology and Bioengineering
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   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0006-3592 - ISSN (Online) 1097-0290
   Published by John Wiley and Sons Homepage  [1579 journals]
  • 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
    • 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
  • Cover Image, Volume 114, Number 11, November 2017
    • Authors: Catherine B. Matthews; Chapman Wright, Angel Kuo, Noelle Colant, Matthew Westoby, J. Christopher Love
      Abstract: Cover Legend: The cover image, by Catherine B. Matthews et al., is based on the Review Reexamining opportunities for therapeutic protein production in eukaryotic microorganisms,
      DOI 10.1002/bit.26378
      PubDate: 2017-09-26T11:09:43.509352-05:
  • 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
    • 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
  • Formic acid as a secondary substrate for succinic acid production by
           metabolically engineered Mannheimia succiniciproducens
    • Authors: Jung Ho Ahn; Junho Bang, Won Jun Kim, Sang Yup Lee
      Abstract: There has been much effort exerted to reduce one carbon (C1) gas emission to address climate change. As one promising way to more conveniently utilize C1 gas, several technologies have been developed to convert C1 gas into useful chemicals such as formic acid (FA). In this study, systems metabolic engineering was utilized to engineer Mannheimia succiniciproducens to efficiently utilize FA. 13C isotope analysis of M. succiniciproducens showed that FA could be utilized through formate dehydrogenase (FDH) reaction and/or the reverse reaction of pyruvate formate lyase (PFL). However, the naturally favored forward reaction of PFL was found to lower the SA yield from FA. In addition, FA assimilation via FDH was found to be more efficient than the reverse reaction of PFL. Thus, the M. succiniciproducens LPK7 strain, which lacks in pfl, ldh, pta, and ack genes, was selected as a base strain. In silico metabolic analysis confirmed that utilization of FA would be beneficial for the enhanced production of SA and suggested FDH as an amplification target. To find a suitable FDH, four different FDHs from M. succiniciproducens, Methylobacterium extorquens, and Candida boidinii were amplified in LPK7 strain to enhance FA assimilation. High-inoculum density cultivation using 13C labeled sodium formate was performed to evaluate FA assimilation efficiency. Fed-batch fermentations of the LPK7 (pMS3-fdh2 meq) strain was carried out using glucose, sucrose, or glycerol as a primary carbon source and FA as a secondary carbon source. As a result, this strain produced 76.11 g/L SA with the yield and productivity of 1.28 mol/mol and 4.08 g/L/h, respectively, using sucrose and FA as dual carbon sources. The strategy employed here will be similarly applicable in developing microorganisms to utilize FA and to produce valuable chemicals and materials from FA.Systems metabolic engineering was performed to develop Mannheimia succiniciproducens LPK7 (pMS3-fdh2meq) strain (ΔldhA, Δpta-ackA, Δpfl, +fdh2) for enhanced utilization of formic acid. Furthermore, production of succinic acid from formic acid as a secondary substrate was demonstrated by fed-batch fermentation.
      PubDate: 2017-09-19T11:40:24.896534-05:
      DOI: 10.1002/bit.26435
  • 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
    • 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
    • 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
    • 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
  • A dynamic metabolic flux analysis of ABE (acetone-butanol-ethanol)
           fermentation by Clostridium acetobutylicum ATCC 824, with riboflavin as a
    • Authors: Xinhe Zhao; Mayssa Kasbi, Jingkui Chen, Sabine Peres, Mario Jolicoeur
      Abstract: The present study reveals that supplementing sodium acetate (NaAc) strongly stimulates riboflavin production in acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum ATCC 824 with xylose as carbon source. Riboflavin production increased from undetectable concentrations to ∼0.2 g L−1 (0.53 mM) when supplementing 60 mM NaAc. Of interest, solvents production and biomass yield were also promoted with fivefold acetone, 2.6-fold butanol, and 2.4-fold biomass adding NaAc. A kinetic metabolic model, developed to simulate ABE biosystem, with riboflavin production, revealed from a dynamic metabolic flux analysis (dMFA) simultaneous increase of riboflavin (ribA) and GTP (precursor of riboflavin) (PurM) synthesis flux rates under NaAc supplementation. The model includes 23 fluxes, 24 metabolites, and 72 kinetic parameters. It also suggested that NaAc condition has first stimulated the accumulation of intracellular metabolite intermediates during the acidogenic phase, which have then fed the solventogenic phase leading to increased ABE production. In addition, NaAc resulted in higher intracellular levels of NADH during the whole culture. Moreover, lower GTP-to-adenosine phosphates (ATP, ADP, AMP) ratio under NaAc supplemented condition suggests that GTP may have a minor role in the cell energetic metabolism compared to its contribution to riboflavin synthesis.Supplementing culture medium with sodium acetate significantly enhances riboflavin production in ABE fermentation with Clostridium acetobutylicum.
      PubDate: 2017-08-29T15:00:41.387848-05:
      DOI: 10.1002/bit.26393
  • Biosimilars: Key regulatory considerations and similarity assessment tools
    • Authors: Carol F. Kirchhoff; Xiao-Zhuo Michelle Wang, Hugh D. Conlon, Scott Anderson, Anne M. Ryan, Arindam Bose
      Abstract: A biosimilar drug is defined in the US Food and Drug Administration (FDA) guidance document as a biopharmaceutical that is highly similar to an already licensed biologic product (referred to as the reference product) notwithstanding minor differences in clinically inactive components and for which there are no clinically meaningful differences in purity, potency, and safety between the two products. The development of biosimilars is a challenging, multistep process. Typically, the assessment of similarity involves comprehensive structural and functional characterization throughout the development of the biosimilar in an iterative manner and, if required by the local regulatory authority, an in vivo nonclinical evaluation, all conducted with direct comparison to the reference product. In addition, comparative clinical pharmacology studies are conducted with the reference product. The approval of biosimilars is highly regulated although varied across the globe in terms of nomenclature and the precise criteria for demonstrating similarity. Despite varied regulatory requirements, differences between the proposed biosimilar and the reference product must be supported by strong scientific evidence that these differences are not clinically meaningful. This review discusses the challenges faced by pharmaceutical companies in the development of biosimilars. This article is protected by copyright. All rights reserved
      PubDate: 2017-08-26T06:05:39.347722-05:
      DOI: 10.1002/bit.26438
  • Cascading effect in bioprocessing – The impact of mild hypothermia on
           CHO cell behaviour and host cell protein composition
    • Authors: Cher H. Goey; Joshua M.H. Tsang, David Bell, Cleo Kontoravdi
      Abstract: A major challenge in downstream purification of monoclonal antibodies (mAb) is the removal of host cell proteins (HCPs). Previous studies have shown that cell culture decisions significantly impact the HCP content at harvest. However, it is currently unclear how process conditions affect physiological changes in the host cell population, and how these changes, in turn, cascade down to change the HCP profile. We examined how temperature downshift (TDS) to mild hypothermia affects key upstream performance indicators, i.e. antibody titre, HCP concentration and HCP species, across the cell culture decline phase and at harvest through the lens of changes in cellular behaviour. Mild hypothermic conditions introduced on day 5 of fed-batch Chinese hamster ovary (CHO) cell bioreactors resulted in a lower cell proliferation rate but larger percentages of healthier cells across the cell culture decline phase compared to bioreactors maintained at standard physiological temperature. Moreover, the onset of apoptosis was less evident in mild hypothermic cultures. Consequently, mild hypothermic cultures took an extra five days to reach an integral viable cell concentration (IVCC) and antibody yield similar to that of the control at standard physiological temperature. When cell viability dropped below 80%, mild hypothermic cell cultures had a reduced variety of HCP species by 36%, including approximately 44% and 27% lower proteases and chaperones, respectively, despite having similar HCP concentration. This study suggests that TDS may be a good strategy to provide cleaner downstream feedstocks by reducing the variety of HCPs and to maintain product integrity by reducing the number of proteases and chaperones. This article is protected by copyright. All rights reserved
      PubDate: 2017-08-26T06:05:37.890537-05:
      DOI: 10.1002/bit.26437
  • 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
  • Recombinant RNA Polymerase from Geobacillus sp. GHH01 as tool for rapid
           generation of metagenomic RNAs using in vitro technologies
    • Authors: Birhanu M. Kinfu; Maike Köster, Mareike Janus, Volkan Besirlioglu, Michael Roggenbuck, Richard Meurer, Ljubica Vojcic, Martin Borchert, Ulrich Schwaneberg, Jennifer Chow, Wolfgang R. Streit
      Abstract: The exciting promises of functional metagenomics for the efficient discovery of novel biomolecules from nature are often hindered by factors associated with expression hosts. Aiming to shift functional metagenomics to a host independent innovative system, we here report on the cloning, heterologous expression and reconstitution of an RNA polymerase (RNAP) from the thermophilic Geobacillus sp. GHH01 and in vitro transcription thereafter. The five genes coding for RNAP subunits, a house keeping sigma factor and two transcription elongation factors were cloned and over expressed as His6-tagged and/ or tag-free proteins. Purified subunits were reconstituted into a functional polymerase through either the classical method of denaturation and subsequent renaturation or through a new resource and time efficient thermo-reconstitution method which takes advantage of the subunits' temperature stability. Additionally, all subunits were cloned into a single vector system for a co-expression and in vivo reconstitution to the RNAP core enzyme. Both the core and holoenzyme form of the RNAP exhibited a robust transcription activity and were stable up to a temperature of 55°C close to their fullest activity. The Geobacillus RNAP showed a remarkable in vitro transcription profile recognizing DNA template sequences of diverse bacteria and archaea as well as metagenomic samples. Coupled with a subsequent in vitro translation step, this recombinant transcription system could allow a new, clone-free and functional metagenomic screening approach. This article is protected by copyright. All rights reserved
      PubDate: 2017-08-26T05:50:30.563045-05:
      DOI: 10.1002/bit.26436
  • Escherichia coli ‘TatExpress’ strains super-secrete human growth
           hormone into the bacterial periplasm by the Tat pathway
    • Authors: Douglas F. Browning; Kirsty L. Richards, Amber R. Peswani, Jo Roobol, Stephen J. W. Busby, Colin Robinson
      Abstract: Numerous high-value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec-based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super-secreting strains by introducing the strong inducible bacterial promoter, ptac, upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g. W3110 and BL21). This modification significantly improves the Tat-dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1 h induction. TatExpress strains accumulate in excess of 30 mg L−1 periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains. This article is protected by copyright. All rights reserved
      PubDate: 2017-08-26T05:50:22.405238-05:
      DOI: 10.1002/bit.26434
  • Impact of Growth Mode, Phase and Rate on the Metabolic State of the
           Extremely Thermophilic Archaeon Pyrococcus furiosus
    • Authors: Piyum A. Khatibi; Chung-jung Chou, Andrew J. Loder, Jeffrey V. Zurawski, Michael W.W. Adams, Robert M. Kelly
      Abstract: The archaeon Pyrococcus furiosus is emerging as a metabolic engineering platform for production of fuels and chemicals, such that more must be known about this organism's characteristics in bioprocessing contexts. Its ability to grow at temperatures from 70 to greater than 100°C and thereby avoid contamination, offers the opportunity for long duration, continuous bioprocesses as an alternative to batch systems. Towards that end, we analyzed the transcriptome of P. furiosus to reveal its metabolic state during different growth modes that are relevant to bioprocessing. As cells progressed from exponential to stationary phase in batch cultures, genes involved in biosynthetic pathways important to replacing diminishing supplies of key nutrients and genes responsible for the onset of stress responses were up-regulated. In contrast, during continuous culture, the progression to higher dilution rates down-regulated many biosynthetic processes as nutrient supplies were increased. Most interesting was the contrast between batch exponential phase and continuous culture at comparable growth rates (∼0.4 h−1), where over 200 genes were differentially transcribed, indicating among other things, N-limitation in the chemostat and the onset of oxidative stress. The results here suggest that cellular processes involved in carbon and electron flux in P. furiosus were significantly impacted by growth mode, phase and rate, factors that need to be taken into account when developing successful metabolic engineering strategies. This article is protected by copyright. All rights reserved
      PubDate: 2017-08-25T06:05:27.228806-05:
      DOI: 10.1002/bit.26408
  • Kinetics based reaction optimization of enzyme catalysed reduction of
           formaldehyde to methanol with synchronous cofactor regeneration
    • Authors: Fauziah Marpani; Zsuzsa Sárossy, Manuel Pinelo, Anne S. Meyer
      Abstract: Enzymatic reduction of carbon dioxide (CO2) to methanol (CH3OH) can be accomplished using a designed set-up of three oxidoreductases utilizing reduced pyridine nucleotide (NADH) as cofactor for the reducing equivalents electron supply. For this enzyme system to function efficiently a balanced regeneration of the reducing equivalents during reaction is required. Herein, we report the optimization of the enzymatic conversion of formaldehyde (CHOH) to CH3OH by alcohol dehydrogenase, the final step of the enzymatic redox reaction of CO2 to CH3OH, with kinetically synchronous enzymatic cofactor regeneration using either glucose dehydrogenase (System I) or xylose dehydrogenase (System II). A mathematical model of the enzyme kinetics was employed to identify the best reaction set-up for attaining optimal cofactor recycling rate and enzyme utilization efficiency. Targeted process optimization experiments were conducted to verify the kinetically modelled results. Repetitive reaction cycles were shown to enhance the yield of CH3OH, increase the total turnover number (TTN) and the biocatalytic productivity rate (BPR) value for both system I and II whilst minimizing the exposure of the enzymes to high concentrations of CHOH. System II was found to be superior to System I with a yield of 8 mM CH3OH, a TTN of 160 and BPR of 24 μmol CH3OH/U·h during 6 hours of reaction. The study demonstrates that an optimal reaction set-up could be designed from rational kinetics modelling to maximize the yield of CH3OH, whilst simultaneously optimizing cofactor recycling and enzyme utilization efficiency. This article is protected by copyright. All rights reserved
      PubDate: 2017-08-23T07:15:24.205539-05:
      DOI: 10.1002/bit.26405
  • Dynamic Monitoring of p53 Translocation to Mitochondria for the Analysis
           of Specific Inhibitors Using Luciferase-fragment Complementation
    • Authors: Natsumi Noda; Raheela Awais, Robert Sutton, Muhammad Awais, Takeaki Ozawa
      Abstract: Intracellular protein translocation plays a pivotal role in regulating complex biological processes, including cell death. The tumor suppressor p53 is a transcription factor activated by DNA damage and oxidative stress that also translocates from the cytosol into the mitochondrial matrix to facilitate necrotic cell death. However, specific inhibitors of p53 mitochondrial translocation are largely unknown. To explore the inhibitors of p53, we developed a bioluminescent probe to monitor p53 translocation from cytosol to mitochondria using luciferase fragment complementation assays. The probe is composed of a novel pair of luciferase fragments, the N-terminus of green click beetle luciferase CBG68 (CBGN) and multiple-complement luciferase fragment (McLuc1). The combination of luciferase fragments showed significant luminescence intensity and high signal-to-background ratio. When the p53 connected with McLuc1 translocates from cytosol into mitochondrial matrix, CBGN in mitochondrial matrix enables to complement with McLuc1, resulting in the restoration of the luminescence. The luminescence intensity was significantly increased under hydrogen peroxide-induced oxidative stress following the complementation of CBGN and McLuc1. Pifithrin-µ, a selective inhibitor of p53 mitochondrial translocation, prevented the mitochondrial translocation of the p53 probe in a concentration-dependent manner. Furthermore, the high luminescence intensity made it easier to visualize the p53 translocation at a single cell level under a bioluminescence microscope. This p53 mitochondrial translocation assay is a new tool for high-throughput screening to identify novel p53 inhibitors, which could be developed as drugs to treat diseases in which necrotic cell death is a major contributor. This article is protected by copyright. All rights reserved
      PubDate: 2017-08-19T06:25:29.174431-05:
      DOI: 10.1002/bit.26407
  • Codelivery of Anti-cancer Agents via Double-walled Polymeric
           Microparticles/ Injectable Hydrogel: A Promising Approach for Treatment of
           Triple Negative Breast Cancer
    • Authors: Pooya Davoodi; Wei Cheng Ng, Madapusi P. Srinivasan, Chi-Hwa Wang
      Abstract: Triple negative breast cancer (TNBC) is an aggressive sub-type of breast cancer that rarely responds to conventional chemotherapy. Therefore, novel agents or new routes need to be developed to improve treatment efficacy and diminish severe side-effects of anti-cancer agents in TNBC patients. This study explores a novel localized co-delivery platform with potential application against TNBC. Uniform core-shell microparticles encapsulating cisplatin (Cis-DDP) and paclitaxel (PTX) are fabricated using coaxial electrohydrodynamic atomization technique and subsequently are embedded into an injectable hydrogel. The hydrogel provides an additional diffusion barrier against Cis-DDP and confines premature release of drugs. In addition, the hydrogel can provide a versatile tool for retaining particles in the tumor resected cavity during the injection following debulking surgery and prevent surgical site infection due to its inherent antibacterial properties. The combination of Cis-DDP and PTX demonstrates a synergistic effect against MDA-MB-231 cell line assigned to three different mechanisms of action, including denaturation of DNA strands, stabilization of microtubules, and amplification of intracellular reactive oxygen species (ROS) and activation of caspase-3 pathways. The results show a significant accumulation of mitochondrial ROS insults in cells upon treatment that consequently causes programmed cells death. The performance of microparticles/hydrogel carrier is evaluated against three-dimensional MDA-MB-231 (breast cancer) 3D spheroids, where a superior efficacy and a greater reduction in spheroid growth are observed over 14 days, as compared with free-drug treatment. Overall, drug-loaded core-shell microparticles embedded into injectable hydrogel provides a promising strategy to treat aggressive cancers and a modular platform for a broad range of localized multidrug therapies customizable to the cancer type.
      PubDate: 2017-08-19T06:25:26.894597-05:
      DOI: 10.1002/bit.26406
  • 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
  • Comparative analysis of plant-produced, recombinant dimeric IgA against
           cell wall β-glucan of pathogenic fungi
    • Authors: Cristina Capodicasa; Marcello Catellani, Ilaria Moscetti, Carla Bromuro, Paola Chiani, Antonella Torosantucci, Eugenio Benvenuto
      Abstract: Immunoglobulins A (IgA) are crucially involved in protection of human mucosal surfaces from microbial pathogens. In this work, we devised and expressed in plants recombinant chimeric antifungal antibodies (Abs) of isotype A (IgA1, IgA2 and scFvFcA1), derived from a murine mAb directed to the fungal cell wall polysaccharide β-glucan which had proven able to confer protection against multiple pathogenic fungi. All recombinant IgA (rIgA) were expressed and correctly assembled in dimeric form in plants and evaluated for yield, antigen-binding efficiency and antifungal properties in vitro, in comparison with a chimeric IgG1 version. Production yields and binding efficiency to purified β-glucans showed significant variations not only between Abs of different isotypes but also between the different IgA formats. Moreover, only the dimeric IgA1 was able to strongly bind cells of the fungal pathogen C. albicans and to restrain its adhesion to human epithelial cells. Our data indicate that IgG to IgA switch and differences in molecular structure among different rIgA formats can impact expression in plant and biological activity of anti-β-glucans Abs and provide new insights for the design of recombinant IgA as anti-infective immunotherapeutics, whose potential is still poorly investigated. This article is protected by copyright. All rights reserved
      PubDate: 2017-08-19T06:20:37.634006-05:
      DOI: 10.1002/bit.26403
  • CRISPRi repression of nonhomologous end-joining for enhanced genome
           engineering via homologous recombination in Yarrowia lipolytica
    • Authors: Cory Schwartz; Keith Frogue, Adithya Ramesh, Joshua Misa, Ian Wheeldon
      Abstract: In many organisms of biotechnological importance precise genome editing is limited by inherently low homologous recombination (HR) efficiencies. A number of strategies exist to increase the effectiveness of this native DNA repair pathway; however, most strategies rely on permanently disabling competing repair pathways, thus reducing an organism's capacity to repair naturally occurring double strand breaks. Here, we describe a CRISPR interference (CRISPRi) system for gene repression in the oleochemical-producing yeast Yarrowia lipolytica. By using a multiplexed sgRNA targeting strategy, we demonstrate efficient repression of 8 out of 9 targeted genes to enhance HR. Strains with nonhomologous end-joining repressed were shown to have increased rates of HR when transformed with a linear DNA fragment with homology to a genomic locus. With multiplexed targeting of KU70 and KU80, and enhanced repression with Mxi1 fused to deactivated Cas9 (dCas9), rates of HR as high as 90% were achieved. The developed CRISPRi system enables enhanced HR in Y. lipolytica without permanent genetic knockouts and promises to be a potent tool for other metabolic engineering, synthetic biology, and functional genomics studies. This article is protected by copyright. All rights reserved
      PubDate: 2017-08-19T06:20:32.62768-05:0
      DOI: 10.1002/bit.26404
  • Recombinant Lactococcus lactis for efficient conversion of cellodextrins
           into L-lactic acid
    • Authors: Chiara Gandini; Loredana Tarraran, Denis Kalemasi, Enrica Pessione, Roberto Mazzoli
      Abstract: Lactic acid bacteria (LAB) are among the most interesting organisms for industrial processes with a long history of application as food starters and biocontrol agents, and an underexploited potential for biorefineries converting biomass into high-value compounds. Lactic acid (LA), their main fermentation product, is among the most requested chemicals owing to its broad range of applications. Notably, LA polymers, i.e., polylactides, have high potential as biodegradable substitutes of fossil-derived plastics. However, LA production by LAB fermentation is currently too expensive for polylactide to be cost-competitive with traditional plastics. LAB have complex nutritional requirements and cannot ferment inexpensive substrates such as cellulose. Metabolic engineering could help reduce such nutritional requirements and enable LAB to directly ferment low-cost polysaccharides.Here, we engineered a Lactococcus lactis strain which constitutively secretes a β-glucosidase and an endoglucanase. The recombinant strain can grow on cellooligosaccharides up to at least cellooctaose and efficiently metabolizes them to L-LA in single-step fermentation. This is the first report of a LAB able to directly metabolize cellooligosaccharides longer that cellohexaose and a significant step towards cost-sustainable consolidated bioprocessing of cellulose into optically pure LA. This article is protected by copyright. All rights reserved
      PubDate: 2017-08-12T05:40:24.680384-05:
      DOI: 10.1002/bit.26400
  • Mechanisms of Nitrous Oxide (N2O) Formation and Reduction in Denitrifying
    • Authors: Fabrizio Sabba; Cristian Picioreanu, Robert Nerenberg
      Abstract: Nitrous oxide (N2O) is a potent greenhouse gas that can be formed in wastewater treatment processes by ammonium oxidizing and denitrifying microorganisms. While N2O emissions from suspended growth systems have been extensively studied, and some recent studies have addressed emissions from nitrifying biofilms, much less is known about N2O emissions from denitrifying biofilm processes. This research used modeling to evaluate the mechanisms of N2O formation and reduction in denitrifying biofilms. The kinetic model included formation and consumption of key denitrification species, including nitrate (NO3-), nitrite (NO2-), nitric oxide (NO), and N2O. The model showed that, in presence of excess of electron donor, denitrifying biofilms have two distinct layers of activity: an outer layer where there is net production of N2O and an inner layer where there is net consumption. The presence of oxygen (O2) had an important effect on N2O emission from suspended growth systems, but a smaller effect on biofilm systems. The effects of NO3- and O2 differed significantly based on the biofilm thickness. Overall, the effects of biofilm thickness and bulk substrate concentrations on N2O emissions are complex and not always intuitive. A key mechanism for denitrifying biofilms is the diffusion of N2O and other intermediates from one zone of the biofilm to another. This leads to zones of N2O formation or consumption transformations that would not exist in suspended growth systems. This article is protected by copyright. All rights reserved
      PubDate: 2017-08-07T04:35:55.423068-05:
      DOI: 10.1002/bit.26399
  • Alginate-gelatin encapsulation of human endothelial cells promoted
           angiogenesis in in vivo and in vitro milieu
    • Authors: Sorour Nemati; Aysa Rezabakhsh, Ali Baradar Khoshfetrat, Alireza Nourazarian, Çığır Biray Avci, Bakiye Goker Bagca, Hamed Alizadeh Sardroud, Majid Khaksar, Mahdi Ahmadi, Aref Delkhosh, Emel Sokullu, Reza Rahbarghazi
      Abstract: Up to present, many advantages have been achieved in the field of cell-based therapies by applying sophisticated methodologies and delivery approaches. Microcapsules are capable to provide safe microenvironment for cells during transplantation in a simulated physiological 3D milieu. Here, we aimed to investigate the effect of alginate-gelatin encapsulation on angiogenic behavior of human endothelial cells over a period of 5 days.Human umbilical vein endothelial cells were encapsulated by alginate-gelatin substrate and incubated for 5 days. MTT and autophagy PCR array analysis were used to monitor cell survival rate. For in vitro angiogenesis analysis, cell distribution of Tie-1, Tie-2, VEGFR-1 and VEGFR-2 were detected by ELISA. In addition to in vitro tubulogenesis assay, we monitored the expression of VE-cadherin by western blotting. The migration capacity of encapsulated HUVECs was studied by measuring MMP-2 and MMP-9 via gelatin zymography. The in vivo angiogenic potential of encapsulated HUVECs was analyzed in immune-compromised mouse implant model during 7 days post-transplantation.We demonstrated that encapsulation promoted HUVECs cell survival and proliferation. Compared to control, no significant differences were observed in autophagic status of encapsulated cells (p > 0.05). The level of Tie-1, Tie-2, VEGFR-1 and VEGFR-2 were increased, but did not reach to significant levels. Encapsulation decreased MMP-2, -9 activity and increased the VE-cadherin level in enclosed cells (p 
      PubDate: 2017-08-07T04:31:14.78254-05:0
      DOI: 10.1002/bit.26395
  • Combination of Traditional Mutation and Metabolic Engineering to Enhance
           Ansamitocin P-3 Production in Actinosynnema pretiosum
    • Authors: Zhi-Qiang Du; Yuan Zhang, Zhi-Gang Qian, Han Xiao, Jian-Jiang Zhong
      Abstract: Ansamitocin P-3 (AP-3) is a maytansinoid with its most compelling antitumor activity, however, the low production titer of AP-3 greatly restricts its wide commercial application. In this work, a combinatorial approach including random mutation and metabolic engineering was conducted to enhance AP-3 biosynthesis in Actinosynnema pretiosum. Firstly, a mutant strain M was isolated by N-methyl-N'-nitro-N-nitrosoguanidine mutation, which could produce AP-3 almost 3-fold that of wild type in 48 deep-well plates. Then, by overexpressing key biosynthetic genes asmUdpg and asm13-17 in the M strain, a further 60% increase of AP-3 production in 250-mL shake flasks was achieved in the engineered strain M-asmUdpg:asm13-17 compared to the M strain, and its maximum AP-3 production reached 582.7 mg/L, which is the highest as ever reported. Both the gene transcription levels and intracellular intermediate concentrations in AP-3 biosynthesis pathway were significantly increased in the M and M-asmUdpg:asm13-17 during fermentation compared to the wild type. The good fermentation performance of the engineered strain was also confirmed in a lab-scale bioreactor. This work demonstrated that combination of random mutation and metabolic engineering could promote AP-3 biosynthesis and might be helpful for increasing the production of other industrially important secondary metabolites. This article is protected by copyright. All rights reserved
      PubDate: 2017-08-07T04:31:10.523571-05:
      DOI: 10.1002/bit.26396
  • 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
  • Controlling Localization of E. coli Populations Using a Two-Part Synthetic
           Motility Circuit: An Accelerator and Brake
    • Authors: Ryan McKay; Pricila Hauk, Hsuan-Chen Wu, Alex Eli Pottash, Wu Shang, Jessica Terrell, Gregory F. Payne, William E. Bentley
      Abstract: Probiotics, whether taken as capsules or consumed in foods, have been regarded as safe for human use by regulatory agencies. Being living cells, they serve as “tunable” factories for the synthesis of a vast array of beneficial molecules. The idea of reprogramming probiotics to act as controllable factories, producing potential therapeutic molecules under user-specified conditions, represents a new and powerful concept in drug synthesis and delivery. Probiotics that serve as drug delivery vehicles pose several challenges, one being targeting (similar to nanoparticle approaches). Here, we employ synthetic biology to control swimming directionality in a process referred to as “pseudotaxis”. E. coli, absent the motility regulator cheZ, swim sporadically, missing the traditional “run” in the run:tumble swimming paradigm. Upon introduction of cheZ in trans and its signal-generated upregulation, engineered bacteria can be “programmed” to swim towards the source of the chemical cue. Here, engineered cells that encounter sufficient levels of the small signal molecule pyocyanin, produce an engineered CheZ and swim with programmed directionality. By incorporating a degradation tag at the C-terminus of CheZ, the cells stop running when they exit spaces containing pyocyanin. That is, the engineered CheZ modified with a C-terminal extension derived from the putative DNA-binding transcriptional regulator YbaQ (RREERAAKKVA) is consumed by the ClpXP protease machine at a rate sufficient to “brake” the cells when pyocyanin levels are too low. Through this process, we demonstrate that over time, these engineered E. coli accumulate in pyocyanin-rich locales. We suggest that such approaches may find utility in engineering probiotics so that their beneficial functions can be focused in areas of principal benefit. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-29T06:35:34.133663-05:
      DOI: 10.1002/bit.26391
  • Metabolic engineering of isopropyl alcohol-producing Escherichia coli
           strains with 13C-metabolic flux analysis
    • Authors: Nobuyuki Okahashi; Fumio Matsuda, Katsunori Yoshikawa, Tomokazu Shirai, Yoshiko Matsumoto, Mitsufumi Wada, Hiroshi Shimizu
      Abstract: Metabolic engineering of isopropyl alcohol (IPA)-producing E. coli strains was conducted along with 13C-metabolic flux analysis (MFA). A metabolically engineered E. coli strain expressing the adc gene derived from Clostridium acetobutylicum and the IPADH gene from C. beijerinckii did not produce IPA during its exponential growth phase in the aerobic batch culture. 13C-MFA was carried out, and revealed a deficiency in NADPH regeneration for IPA production in growth phase. Based on these findings, we used nitrogen-starved culture conditions to reduce NADPH consumption for biomass synthesis. As a result, IPA yield was increased to 20% mol/mol glucose. 13C-MFA revealed that the relative flux levels through the oxidative pentose phosphate (PP) pathway and the TCA cycle were elevated in nitrogen-starved condition relative to glucose uptake rate. To prevent CO2 release in the 6-phosphogluconate dehydrogenase (6PGDH) reaction, metabolism of this E. coli strain was further engineered to redirect glycolytic flux to the glucose 6-phosphate dehydrogenase (G6PDH) and Entner-Doudoroff (ED) pathway. IPA yield of 55% mol/mol glucose was achieved by combining the nitrogen-starved culture condition with the metabolic redirection. The 13C-MFA data and intracellular NADPH levels obtained under these IPA production conditions revealed linear correlations between the specific IPA production rate and NADPH concentration, as well as between IPA yield and the pyruvate dehydrogenase (PDH) flux. Our results showed that 13C-MFA is a helpful tool for metabolic engineering studies, and that further improvement in IPA production by E. coli may be achieved by fine-tuning the cofactor ratio and concentrations, as well as optimizing the metabolic pathways and culture conditions. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-29T06:35:24.261834-05:
      DOI: 10.1002/bit.26390
  • Bidirectional reporter assay using HAL promoter and TOPFLASH improves
           specificity in high-throughput screening of Wnt inhibitors
    • Authors: Kiyoshi Yamaguchi; Chi Zhu, Tomoyuki Ohsugi, Yuko Yamaguchi, Tsuneo Ikenoue, Yoichi Furukawa
      Abstract: Constitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. Cell-based assays using synthetic TCF/LEF (T-cell factor/lymphoid enhancer factor) reporters, as readouts of β-catenin/TCF-dependent transcriptional activity, have contributed greatly to the discovery of small molecules that modulate Wnt signaling. In the present study, we report a novel screening method, called a bidirectional dual reporter assay. Integrated transcriptome analysis identified a histidine ammonia-lyase gene (HAL) that was negatively regulated by β-catenin/TCF-dependent transcriptional activity. We leveraged a promoter region of the HAL gene as another transcriptional readout of Wnt signaling. Cells stably expressing both an optimized HAL reporter and the TCF/LEF reporter enabled bidirectional reporter activities in response to Wnt signaling. Increased HAL reporter activity and decreased TCF/LEF reporter activity were observed simultaneously in the cells when β-catenin/TCF7L2 was inhibited. Notably, this method could decrease the number of false positives observed when screening an inhibitor library compared with the conventional TCF/LEF assay. We found that Brefeldin A, a disruptor of the Golgi apparatus, inhibited the Wnt/β-catenin signaling pathway. The utility of our system could be expanded to examine other disease-associated pathways beyond the Wnt/β-catenin signaling pathway. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-29T06:30:43.923551-05:
      DOI: 10.1002/bit.26394
  • NMR Investigation of Water Diffusion in different Biofilm Structures
    • Authors: Maria P. Herrling; Jessica Weisbrodt, Catherine M. Kirkland, Nathan H. Williamson, Susanne Lackner, Sarah L. Codd, Joseph D. Seymour, Gisela Guthausen, Harald Horn
      Abstract: Mass transfer in biofilms is determined by diffusion. Different mostly invasive approaches have been used to measure diffusion coefficients in biofilms, however data on heterogeneous biomass under realistic conditions is still missing. To non-invasively elucidate fluid-structure-interactions in complex multispecies biofilms pulsed field gradient-nuclear magnetic resonance (PFG-NMR) was applied to measure the water diffusion in five different types of biomass aggregates: one type of sludge flocs, two types of biofilm, and two types of granules. Data analysis is an important issue when measuring heterogeneous systems and is shown to significantly influence the interpretation and understanding of water diffusion. With respect to numerical reproducibility and physico-chemical interpretation, different data processing methods were explored: (bi)-exponential data analysis and the Γ distribution model. Furthermore, the diffusion coefficient distribution in relation to relaxation was studied by D-T2 maps obtained by 2D inverse Laplace transform (2D ILT). The results show that the effective diffusion coefficients for all biofilm samples ranged from 0.36 to 0.96 relative to that of water. NMR diffusion was linked to biofilm structure (e.g. biomass density, organic and inorganic matter) as observed by magnetic resonance imaging and to traditional biofilm parameters: Diffusion was most restricted in granules with compact structures, and fast diffusion was found in heterotrophic bio-films with fluffy structures. The effective diffusion coefficients in the biomass were found to be broadly distributed because of internal biomass heterogeneities, such as gas bubbles, precipitates, and locally changing biofilm densities. Thus, estimations based on biofilm bulk properties in multispecies systems can be overestimated and mean diffusion coefficients might not be sufficiently informative to describe mass transport in biofilms and the near bulk. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-29T06:30:24.788066-05:
      DOI: 10.1002/bit.26392
  • Preselection of recombinant gene integration sites enabling high
           transcription rates in CHO cells using alternate start codons and
           Recombinase Mediated Cassette Exchange
    • Authors: Martina Baumann; Elisabeth Gludovacz, Natalie Sealover, Scott Bahr, Henry George, Nan Lin, Kevin Kayser, Nicole Borth
      Abstract: Site-specific Recombinase Mediated Cassette Exchange (RMCE) enables the transfer of the gene of interest (GOI) into pre-selected genomic locations with defined expression properties. For the generation of recombinant production cell lines this has the advantage that screening for high transcription rates at the genome integration site would be required only once, with the possibility to reuse the selected site for new products. Here we describe a strategy that aims at the selection of transcriptionally active genome integration sites in Chinese Hamster Ovary (CHO) cells by using alternate start codons in the surface reporter protein CD4, in combination with FACS sorting for high expressers. The alternate start codon reduces the translation initiation efficiency and allows sorting for CHO cells with the highest transcription rates, while RMCE enables the subsequent exchange of the CD4 against the GOI. We have shown that sorted cell pools with the CD4 reporter gene containing the alternate start codon CTG lead to higher GFP signals and higher antibody titers upon RMCE as compared to cell pools containing the ATG start codon of the CD4 reporter. Despite the absence of any subcloning step, the final cell pool contained the CD4 gene in a single genome integration site. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-22T05:50:39.05428-05:0
      DOI: 10.1002/bit.26388
  • Development of aptamers against unpurified proteins
    • Authors: Shinichi Goto; Kaori Tsukakoshi, Kazunori Ikebukuro
      Abstract: SELEX (Systematic Evolution of Ligands by EXponential enrichment) has been widely used for the generation of aptamers against target proteins. However, its requirement for pure target proteins remains a major problem in aptamer selection, as procedures for protein purification from crude bio-samples are not only complicated but also time and labour consuming. This is because native proteins can be found in a large number of diverse forms because of posttranslational modifications and their complicated molecular conformations. Moreover, several proteins are difficult to purify owing to their chemical fragility and/or rarity in native samples. An alternative route is the use of recombinant proteins for aptamer selection, because they are homogenous and easily purified. However, aptamers generated against recombinant proteins produced in prokaryotic cells may not interact with the same proteins expressed in eukaryotic cells because of posttranslational modifications. Moreover, to date recombinant proteins have been constructed for only a fraction of proteins expressed in the human body. Therefore, the demand for advanced SELEX methods not relying on complicated purification processes from native samples or recombinant proteins is growing. This review article describes several such techniques that allow researchers to directly develop an aptamer from various unpurified samples, such as whole cells, tissues, serum, and cell lysates. The key advantages of advanced SELEX are that it does not require a purification process from a crude bio-sample, maintains the functional states of target proteins, and facilitates the development of aptamers against unidentified and uncharacterized proteins in unpurified biological samples. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-22T05:50:33.03994-05:0
      DOI: 10.1002/bit.26389
  • A Review on Comparative Mechanistic Studies of Antimicrobial Peptides
           against Archaea
    • Authors: Kyriakos G. Varnava; Ron S. Ronimus, Vijayalekshmi Sarojini
      Abstract: Archaea was until recently considered as a third domain of life in addition to bacteria and eukarya but recent studies support the existence of only two superphyla (bacteria and archaea). The fundamental differences between archaeal, bacterial and eukaryal cells are probably the main reasons for the comparatively lower susceptibility of archaeal strains to current antimicrobial agents. The possible emerging pathogenicity of archaea and the role of archaeal methanogens in methane emissions, a potent greenhouse gas, has led many researchers to examine the sensitivity patterns of archaea and make attempts to find agents that have significant anti-archaeal activity. Even though antimicrobial peptides (AMPs) are well known with several published reviews concerning their mode of action against bacteria and eukarya, to our knowledge, to date no reviews are available that focus on the action of these peptides against archaea. Herein we present a review on all the peptides that have been tested against archaea. In addition, in an attempt to shed more light on possible future work that needs to be performed we have included a brief overview of the chemical characteristics, spectrum of activity and the known mechanism of action of each of these peptides against bacteria and/or fungi. We also discuss the nature of and key physiological differences between Archaea, Bacteria and Eukarya that are relevant to the development of anti-archaeal peptides. Despite our relatively limited knowledge about archaea, available data suggest that AMPs have an even broader spectrum of activity than currently recognized. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-22T05:50:24.813697-05:
      DOI: 10.1002/bit.26387
  • Opportunities and Challenges of Real-Time Release Testing in
           Biopharmaceutical Manufacturing
    • Authors: Mo Jiang; Kristen Severson, J. Christopher Love, Helena Madden, Patrick Swann, Li Zang, Richard D. Braatz
      Abstract: Real-time release testing (RTRT) is defined as “the ability to evaluate and ensure the quality of in-process and/or final drug product based on process data, which typically includes a valid combination of measured material attributes and process controls” (ICH Q8(R2)). This article discusses sensors (process analytical technology, PAT) and control strategies that enable RTRT for the spectrum of critical quality attributes (CQAs) in biopharmaceutical manufacturing. Case studies from the small-molecule and biologic pharmaceutical industry are described to demonstrate how RTRT can be facilitated by integrated manufacturing and multivariable control strategies to ensure the quality of products. RTRT can enable increased assurance of product safety, efficacy, and quality – with improved productivity including faster release and potentially decreased costs – all of which improve the value to patients. To implement a complete RTRT solution, biologic drug manufacturers need to consider the special attributes of their industry, particularly sterility and the measurement of viral and microbial contamination. Continued advances in on-line and in-line sensor technologies are key for the biopharmaceutical manufacturing industry to achieve the potential of RTRT. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-15T06:20:34.280285-05:
      DOI: 10.1002/bit.26383
  • Generation of apoptosis-resistant HEK293 cells with CRISPR/Cas mediated
           quadruple gene knockout for improved protein and virus production
    • Authors: Weifeng Zhang; Dan Xiao, Linlin Shan, Junli Zhao, Qinwen Mao, Haibin Xia
      Abstract: Apoptosis has important functions during pathophysiologic processes. However, from a biopharmaceutical point of view, active apoptosis of host cells is undesirable during viral packaging or protein expression, because it decreases the efficiency of viral or protein production. Here we used the CRISPR/Cas technique to knock out four pro-apoptotic genes, Caspase3, Caspase6, Caspase7 and AIF1, in HEK293 cells, and successfully produced an apoptosis-resistant cell line. Furthermore, this cell line showed higher expression levels of pro-apoptotic proteins and higher packaging efficiency for the virus carrying these proteins than control HEK293 cells. This study not only produced an apoptosis-resistant cell line that is useful in producing apoptosis-inducing proteins or viruses expressing these proteins, but also provides a methodology to build other apoptosis-resistant cell lines. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-15T06:20:32.773199-05:
      DOI: 10.1002/bit.26382
  • Coupling the pretreatment and hydrolysis of lignocellulosic biomass by the
           expression of beta-xylosidases
    • Authors: Lucía Martín Pérez; Laura Benítez Casanova, Antonio Javier Moreno Pérez, Dolores Pérez Gómez, Sandra Gavaldá Martín, Laura Ledesma-García, Noelia Valbuena, Bruno Díez García, Francisco Manuel Reyes-Sosa
      Abstract: Thermochemical pretreatment and enzymatic hydrolysis are the areas contributing most to the operational costs of second generation ethanol in lignocellulosic biorefineries. The improvement of lignocellulosic enzyme cocktails has been significant in the recent years. Although the needs for the reduction of the energy intensity and chemical consumption in the pretreatment step are well known, the reduction of the severity of the process strongly affects the enzymatic hydrolysis yield. To explore the formulation requirements of the well known cellulolytic cocktail from Myceliophthora thermophila on mild pretreated raw materials, this cocktail was tested on steam exploded corn stover without acid impregnation. The low hemicellulose yield and significant accumulation of xylobiose compared with the standard pretreated material obtained with dilute acid impregnation evidenced a clear limitation in the conversion of xylan to xylose. In order to complement the beta-xylosidase limitation, a selection of enzymes was expressed and tested in this fungus. A controlled expression of xylosidases from Aspergillus nidulans, Aspergillus fumigatus and Fusarium oxysporum allowed recovering hemicellulose yields reached with standard acid treated material. The results underline the need of parallel development of the pretreatment process with the optimization of the formulation of the enzymatic cocktails. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-15T06:20:30.195008-05:
      DOI: 10.1002/bit.26386
  • ROS Mediated Selection for Increased NADPH Availability in Escherichia
    • Authors: T. Steele Reynolds; Colleen M. Courtney, Keesha E. Erickson, Lisa M. Wolfe, Anushree Chatterjee, Prashant Nagpal, Ryan T. Gill
      Abstract: The economical production of chemicals and fuels by microbial processes remains an intense area of interest in biotechnology. A key limitation in such efforts concerns the availability of key co-factors, in this case NADPH, required for target pathways. Many of the strategies pursued for increasing NADPH availability in Escherichia coli involve manipulations to the central metabolism, which can create redox imbalances and overall growth defects. In this study we used a reactive oxygen species based selection to search for novel methods of increasing NADPH availability. We report a loss of function mutation in the gene hdfR appears to increase NADPH availability in E. coli. Additionally, we show this excess NADPH can be used to improve the production of 3HP in E. coli. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-15T06:20:28.382192-05:
      DOI: 10.1002/bit.26385
  • Quantitative intracellular flux modeling and applications in
           biotherapeutic development and production using CHO cell cultures
    • Authors: Zhuangrong Huang; Dong-Yup Lee, Seongkyu Yoon
      Abstract: Chinese hamster ovary (CHO) cells have been widely used for producing many recombinant therapeutic proteins. Constraint-based modeling, such as flux balance analysis (FBA) and metabolic flux analysis (MFA), has been developing rapidly for the quantification of intracellular metabolic flux distribution at a systematic level. Such methods would produce detailed maps of flows through metabolic networks, which contribute significantly to better understanding of metabolism in cells. Although these approaches have been extensively established in microbial systems, their application to mammalian cells is sparse. This review brings together the recent development of constraint-based models and their applications in CHO cells. The further development of constraint-based modeling approaches driven by multi-omics datasets is discussed, and a framework of potential modeling application in cell culture engineering is proposed. Improved cell culture system understanding will enable robust developments in cell line and bioprocess engineering thus accelerating consistent process quality control in biopharmaceutical manufacturing. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-15T06:20:25.890158-05:
      DOI: 10.1002/bit.26384
  • To Be Certain About the Uncertainty: Bayesian Statistics for 13C Metabolic
           Flux Analysis
    • Authors: Axel Theorell; Samuel Leweke, Wolfgang Wiechert, Katharina Nöh
      Abstract: 13C Metabolic Fluxes Analysis (13C MFA) remains to be the most powerful approach to determine intracellular metabolic reaction rates. Decisions on strain engineering and experimentation heavily rely upon the certainty with which these fluxes are estimated. For uncertainty quantification, the vast majority of 13C MFA studies relies on confidence intervals from the paradigm of Frequentist statistics. However, it is well known that the confidence intervals for a given experimental outcome are not uniquely defined. As a result, confidence intervals produced by different methods can be different, but nevertheless equally valid. This is of high relevance to 13C MFA, since practitioners regularly use three different approximate approaches for calculating confidence intervals. By means of a computational study with a realistic model of the central carbon metabolism of E. coli, we provide strong evidence that confidence intervals used in the field depend strongly on the technique with which they were calculated and, thus, their use leads to misinterpretation of the flux uncertainty. In order to provide a better alternative to confidence intervals in 13C MFA, we demonstrate that credible intervals from the paradigm of Bayesian statistics give more reliable flux uncertainty quantifications which can be readily computed with high accuracy using Markov Chain Monte Carlo. In addition, the widely applied chi-square test, as a means of testing whether the model reproduces the data, is examined closer. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-11T06:25:40.556109-05:
      DOI: 10.1002/bit.26379
  • Metabolic modelling to identify engineering targets for Komagataella
           phaffii: The effect of biomass composition on gene target identification
    • Authors: Ayca Cankorur-Cetinkaya; Duygu Dikicioglu, Stephen G. Oliver
      Abstract: Genome-scale metabolic models are valuable tools for the design of novel strains of industrial microorganisms, such as Komagataella phaffii (syn. Pichia pastoris). However, as is the case for many industrial microbes, there is no executable metabolic model for K. phaffiii that confirms to current standards by providing the metabolite and reactions IDs, to facilitate model extension and reuse, and gene-reaction associations to enable identification of targets for genetic manipulation. In order to remedy this deficiency, we decided to reconstruct the genome-scale metabolic model of K. phaffii by reconciling the extant models and performing extensive manual curation in order to construct an executable model (Kp.1.0) that conforms to current standards. We then used this model to study the effect of biomass composition on the predictive success of the model. Twelve different biomass compositions obtained from published empirical data obtained under a range of growth conditions were employed in this investigation. We found that the success of Kp1.0 in predicting both gene essentiality and growth characteristics was relatively unaffected by biomass composition. However, we found that biomass composition had a profound effect on the distribution of the fluxes involved in lipid, DNA and steroid biosynthetic processes, cellular alcohol metabolic process and oxidation-reduction process. Further, we investigated the effect of biomass composition on the identification of suitable target genes for strain development. The analyses revealed that around 40% of the predictions of the effect of gene overexpression or deletion changed depending on the representation of biomass composition in the model. Considering the robustness of the in silico flux distributions to the changing biomass representations enables better interpretation of experimental results, reduces the risk of wrong target identification, and so both speeds and improves the process of directed strain development. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-10T02:50:20.381487-05:
      DOI: 10.1002/bit.26380
  • Mechanistic insights into the liquefaction stage of enzyme-mediated
           biomass deconstruction
    • Authors: Timo van der Zwan; Jinguang Hu, Jack N. Saddler
      Abstract: Effective enzyme-mediated viscosity reduction, disaggregation, or ‘liquefaction’, is required to overcome the rheological challenges resulting from the fibrous, hygroscopic nature of lignocellulosic biomass, particularly at the high solids loadings that will be required for an economically viable process. However, the actual mechanisms involved in enzyme-mediated deconstruction, as determined by viscosity or yield stress reduction, have yet to be fully resolved. Particle fragmentation, interparticle interaction, material dilution and yield stress rheometry were compared for their ability to quantify enzyme-mediated liquefaction of model and more realistic pretreated biomass substrates. It was apparent that material dilution and particle fragmentation occurred simultaneously and that both mechanisms contributed to viscosity/yield stress reduction. However, their relative importance was dependent on the nature of the biomass substrate. Interparticle interaction and enzyme-mediated changes to these interactions was shown to have a significant effect on slurry rheology. Liquefaction was shown to result from the combined action of material dilution, particle fragmentation, and alteration of interactions at particle surfaces. However, the observed changes in water retention capacity did not correlate with yield stress reduction. The relative importance of each mechanism was significantly influenced by the nature of the biomass substrate and its physicochemical properties. An ongoing challenge is that mechanisms, such as refining, which enhance enzyme accessibility to the cellulosic component of the substrate, are detrimental to slurry rheology and will likely impede enzyme-mediated liquefaction when high substrate concentrations are used. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-10T01:22:57.923928-05:
      DOI: 10.1002/bit.26381
  • Fermentation and purification strategies for the production of betulinic
           acid and its lupane-type precursors in Saccharomyces cerevisiae
    • Authors: Eik Czarnotta; Mariam Dianat, Marcel Korf, Fabian Granica, Juliane Merz, Jérôme Maury, Simo Abdessamad Baallal Jacobsen, Jochen Förster, Birgitta E. Ebert, Lars M. Blank
      Abstract: Microbial production of plant derived, biologically active compounds has the potential to provide economic and ecologic alternatives to existing low productive, plant-based processes. Current production of the pharmacologically active cyclic triterpenoid betulinic acid is realized by extraction from the bark of plane tree or birch. Here, we reengineered the reported betulinic acid pathway into S. cerevisiae and used this novel strain to develop efficient fermentation and product purification methods. Fed-batch cultivations with ethanol excess, using either an ethanol-pulse feed or controlling a constant ethanol concentration in the fermentation medium, significantly enhanced production of betulinic acid and its triterpenoid precursors. The beneficial effect of excess ethanol was further exploited in nitrogen-limited resting cell fermentations, yielding betulinic acid concentrations of 182 mg/L and total triterpenoid concentrations of 854 mg/L, the highest concentrations reported so far. Purification of lupane-type triterpenoids with high selectivity and yield was achieved by solid-liquid extraction without prior cell disruption using polar aprotic solvents such as acetone or ethyl acetate and subsequent precipitation with strong acidsThis study highlights the potential of microbial production of plant derived triterpenoids in S. cerevisiae by combining metabolic and process engineering. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-08T08:51:06.134089-05:
      DOI: 10.1002/bit.26377
  • Quantifying the efficiency of Saccharomyces cerevisiae translocation tags
    • Authors: Amy M. Ehrenworth; Mitchell A. Haines, Amy Wong, Pamela Peralta-Yahya
      Abstract: Compartmentalization of metabolic pathways into organelles of the yeast Saccharomyces cerevisiae has been used to improve chemical production. Pathway compartmentalization aids chemical production by bringing enzymes into close proximity to one another, placing enzymes near key starting metabolites or essential co-factors, increasing the effective concentration of metabolic intermediates, and providing a more suitable chemical environment for enzymatic activity. Although several translocation tags have been used to localize enzymes to different yeast organelles, their translocation efficiencies have not been quantified. Here, we systematically quantify the translocation efficiencies of the ten commonly used S. cerevisiae tags by localizing green fluorescent protein into three yeast organelles: the mitochondrion (4 tags), the vacuole (3 tags), and the peroxisome (3 tags). Further, we investigate whether plasmid copy number or mRNA levels vary with tag translocation efficiency. Quantification efficiencies of S. cerevisiae translocation tags provides an important resource for bioengineering practitioners when choosing a tag to compartmentalize their desired protein. Finally, these efficiencies can be used to determine the percentage of enzyme compartmentalization and, thus, help better quantify effects of compartmentalization on metabolic pathway efficiency. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-08T08:51:00.259213-05:
      DOI: 10.1002/bit.26376
  • Reexamining opportunities for therapeutic protein production in eukaryotic
    • Authors: M. Catherine Bartlett; Chapman Wright, Angel Kuo, Noelle Colant, Matthew Westoby, J. Christopher Love
      Abstract: Antibodies are an important class of therapeutics and are predominantly produced in Chinese Hamster Ovary (CHO) cell lines. While this manufacturing platform is sufficiently productive to supply patient populations of currently approved therapies, it is unclear whether or not the current CHO platform can address two significant areas of need: affordable access to biologics for patients around the globe and production of unprecedented quantities needed for very large populations of patients. Novel approaches to recombinant protein production for therapeutic biologic products may be needed, and might be enabled by non-mammalian expression systems and recent advances in bioengineering. Eukaryotic microorganisms such as fungi, microalgae, and protozoa offer the potential to produce high-quality antibodies in large quantities. In this review, we lay out the current understanding of a wide range of species and evaluate based on theoretical considerations which are best poised to deliver a step change in cost of manufacturing and volumetric productivity within the next decade. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-08T08:40:36.744298-05:
      DOI: 10.1002/bit.26378
  • Combinatorial genome and protein engineering yields monoclonal antibodies
           with hypergalactosylation from CHO cells
    • Authors: Cheng-yu Chung; Qiong Wang, Shuang Yang, Sean A. Ponce, Brian J. Kirsch, Hui Zhang, Michael J. Betenbaugh
      Abstract: One of the key quality attributes of monoclonal antibodies is the glycan pattern and distribution. Two terminal galactose residues typically represent a small fraction of the total glycans from antibodies. However, antibodies with defined glycosylation properties including enhanced galactosylation have been shown to exhibit improved functional properties for these important biomedical modalities. In this study, the disruption of two α-2,3 sialyltransferases (ST3GAL4 and ST3GAL6) from Chinese Hamster Ovary (CHO) cells was combined with protein engineering of the Fc region to generate an IgG containing 80% bigalactosylated and fucosylated (G2F) glycoforms. Expression of the same single amino acid mutant (F241A) IgG in CHO cells with a triple gene knockout of fucosyltransferase (FUT8) plus ST3GAL4 and ST3GAL6 lowered the galactosylation glycoprofile to 65% bigalactosylated G2 glycans. However, overexpression of IgGs with four amino acid substitutions recovered the G2 glycoform composition approximately 80%. Combining genome and protein engineering in CHO cells will provide a new antibody production platform that enables biotechnologists to generate glycoforms with properties tailored for specific in vivo therapeutic and other biomedical applications. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-07T07:00:38.264608-05:
      DOI: 10.1002/bit.26375
  • Small cause, large effect: Structural characterization of cutinases from
           Thermobifida cellulosilytica
    • Authors: Doris Ribitsch; Altijana Hromic, Sabine Zitzenbacher, Barbara Zartl, Caroline Gamerith, Alessandro Pellis, Alois Jungbauer, Andrzej Łyskowski, Georg Steinkellner, Karl Gruber, Rupert Tscheliessnig, Enrique Herrero Acero, Georg M. Guebitz
      Abstract: We have investigated the structures of two native cutinases from Thermobifida cellulosilytica, namely Thc_Cut1 and Thc_Cut2 as well as of two variants, Thc_Cut2_DM (Thc_Cut2_ Arg29Asn_Ala30Val) and Thc_Cut2_TM (Thc_Cut2_Arg19Ser_Arg29Asn_Ala30Val). The four enzymes showed different activities towards the aliphatic polyester poly(lactic acid) (PLLA). The crystal structures of the four enzymes were successfully solved and in combination with Small Angle X-Ray Scattering (SAXS) the structural features responsible for the selectivity difference were elucidated. Analysis of the crystal structures did not indicate significant conformational differences among the different cutinases. However, the distinctive SAXS scattering data collected from the enzymes in solution indicated a remarkable surface charge difference. The difference in the electrostatic and hydrophobic surface properties could explain potential alternative binding modes of the four cutinases on PLLA explaining their distinct activities. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-03T06:04:05.129349-05:
      DOI: 10.1002/bit.26372
  • An efficient model construction strategy to simulate microalgal lutein
           photo-production dynamic process
    • Authors: Ehecatl Antonio del Rio-Chanona; Fabio Fiorelli, Dongda Zhang, Nur rashid Ahmed, Keju Jing, Nilay Shah
      Abstract: Lutein is a high-value bioproduct synthesised by microalga Desmodesmus sp.. It has great potential for the food, cosmetics, and pharmaceutical industries. However, in order to enhance its productivity and to fulfil its ever-increasing global market demand, it is vital to construct accurate models capable of simulating the entire behaviour of the complicated dynamics of the underlying biosystem. To this aim, in this study two highly robust artificial neural networks are designed for the first time. Contrary to conventional artificial neural networks, these networks model the rate of change of the dynamic system, which makes them highly relevant in practice. Different strategies are incorporated into the current research to guarantee the accuracy of the constructed models, which include determining the optimal network structure through a hyper-parameter selection framework, generating significant amounts of artificial data sets by embedding random noise of appropriate size, and rescaling model inputs through standardisation. Based on experimental verification, the high accuracy and great predictive power of the current models for long-term dynamic bioprocess simulation in both real-time and offline frameworks are thoroughly demonstrated. This research, therefore, paves the way to significantly facilitate the future investigation of lutein bioproduction process control and optimisation. In addition, the model construction strategy developed in this research has great potential to be directly applied to other bioprocesses. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-03T06:03:02.796023-05:
      DOI: 10.1002/bit.26373
  • Overflow Metabolism and Growth Cessation in Clostridium thermocellum
           DSM1313 during High Cellulose Loading Fermentations
    • Authors: R. Adam Thompson; Cong T. Trinh
      Abstract: As a model thermophilic bacterium for the production of second-generation biofuels, the metabolism of Clostridium thermocellum has been widely studied. However, most studies have characterized C. thermocellum metabolism for growth at relatively low substrate concentrations. This outlook is not industrially relevant, however, as commercial viability requires substrate loadings of at least 100 g/L cellulosic materials. Recently, a wild-type C. thermocellum DSM1313 was cultured on high cellulose loading batch fermentations and reported to produce a wide range of fermentative products not seen at lower substrate concentrations, opening the door for a more in-depth analysis of how this organism will behave in industrially relevant conditions. In this work, we elucidated the interconnectedness of overflow metabolism and growth cessation in C. thermocellum during high cellulose loading batch fermentations (100 g/L). Metabolic flux and thermodynamic analyses suggested that hydrogen and formate accumulation perturbed the complex redox metabolism and limited conversion of pyruvate to acetyl-CoA conversion, likely leading to overflow metabolism and growth cessation in C. thermocellum. Pyruvate formate lyase (PFL) acts as an important redox valve and its flux is inhibited by formate accumulation. Finally, we demonstrated that manipulation of fermentation conditions to alleviate hydrogen accumulation could dramatically alter the fate of pyruvate, providing valuable insight into process design for enhanced C. thermocellum production of chemicals and biofuels. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-03T06:01:27.587032-05:
      DOI: 10.1002/bit.26374
  • Intracellular characterization of Gag VLP production by transient
           transfection of HEK 293 cells
    • Authors: Laura Cervera; Irene González-Domínguez, Maria Mercedes Segura, Francesc Gòdia
      Abstract: Transient transfection is a fast, flexible, and cost-effective approach to produce biological products. Despite the continued interest in transient transfection, little is known regarding the transfection process at the intracellular level, particularly for complex products, such as virus-like particles (VLPs). The kinetics of PEI-mediated transfection following an established in-house protocol is reported in this work with the aim of characterizing and understanding the complete process leading to VLP generation and identifying important events driving process improvement. For this purpose, DNA/PEI polyplexes' internalization in cells was tracked using Cy3 DNA staining. The production of a fluorescently labeled Gag polyprotein (a Gag-GFP fusion construct that forms fluorescent Gag-VLPs) was monitored by flow cytometry and confocal microscopy, and the VLP concentration in supernatants was measured by fluorometry. DNA/PEI polyplexes have been shown to interact with the cell membrane immediately after polyplex addition to the cell culture. A linear increase in the number of cells expressing the protein is observed during the first 60 minutes of contact between the cells and polyplexes. No additional improvement in the number of cells expressing the protein (up to 60%) or VLP production (up to 1 × 1010 VLPs/mL) is observed with additional contact time between the cells and polyplexes. Polyplexes can be detected in the cytoplasm of transfected cells as early as 1.5 hours post-transfection (hpt) and reach the nucleus approximately 4 hpt. GFP fluorescence is observed homogeneously in the cytoplasm of transfected cells 24 hpt, but generalized VLP budding is not observed by microscopy until 48 hpt. Although all cells have internalized a polyplex soon after transfection, only a fraction of cells (60%) express the fluorescent Gag protein. VLP production kinetics was also studied. Fluorescence in the supernatant (enveloped VLPs) is 40% less than total fluorescence, supernatant plus pellet (free Gag-GFP), indicating that there is a fraction of Gag that remains inside the cells. The maximum VLP concentration in the cell culture supernatant with cell viability>89% was observed at 72 hpt, which was determined to be the optimal harvest time. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-01T07:50:35.831771-05:
      DOI: 10.1002/bit.26367
  • Developing global regression models for metabolite concentration
           prediction regardless of cell line
    • Authors: Silvère André; Sylvain Lagresle, Anthony Da Sliva, Pierre Heimendinger, Zahia Hannas, Éric Calvosa, Ludovic Duponchel
      Abstract: Following the Process Analytical Technology (PAT) of the Food and Drug Administration (FDA), drug manufacturers are encouraged to develop innovative techniques in order to monitor and understand their processes in a better way. Within this framework, it has been demonstrated that Raman spectroscopy coupled with chemometric tools allow to predict critical parameters of mammalian cell cultures in-line and in real time. However, the development of robust and predictive regression models clearly requires many batches in order to take into account inter-batch variability and enhance models accuracy. Nevertheless, this heavy procedure has to be repeated for every new line of cell culture involving many resources. This is why we propose in this paper to develop global regression models taking into account different cell lines. Such models are finally transferred to any culture of the cells involved. This article first demonstrates the feasibility of developing regression models, not only for mammalian cell lines (CHO and HeLa cell cultures), but also for insect cell lines (Sf9 cell cultures). Then global regression models are generated, based on CHO cells, HeLa cells and Sf9 cells. Finally, these models are evaluated considering a fourth cell line(HEK cells). In addition to suitable predictions of glucose and lactate concentration of HEK cell cultures, we expose that by adding a single HEK-cell culture to the calibration set, the predictive ability of the regression models are substantially increased. In this way, we demonstrate that using global models, it is not necessary to consider many cultures of a new cell linein order to obtain accurate models. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-01T07:45:18.769046-05:
      DOI: 10.1002/bit.26368
  • Integrated Gut/Liver Microphysiological Systems Elucidates Inflammatory
           Inter-Tissue Crosstalk
    • Authors: Wen Li Kelly Chen; Collin Edington, Emily Suter, Jiajie Yu, Jeremy J. Velazquez, Jason G. Velazquez, Michael Shockley, Emma M. Large, Raman Venkataramanan, David J. Hughes, Cynthia L. Stokes, David L. Trumper, Rebecca L. Carrier, Murat Cirit, Linda G. Griffith, Douglas A. Lauffenburger
      Abstract: A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut-liver tissue interactions under normal and inflammatory contexts, via an integrative multi-organ platform comprising human liver (hepatocytes and Kupffer cells) and intestinal (enterocyte, goblet cells, and dendritic cells) models. Our results demonstrated long-term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut-liver crosstalk. Moreover, significant non-linear modulation of cytokine responses was observed under inflammatory gut-liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA-seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut-liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut-liver interaction also negatively affected tissue-specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi-tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-01T07:40:31.096633-05:
      DOI: 10.1002/bit.26370
  • Empirical Modeling of T cell Activation Predicts Interplay of Host
           Cytokines and Bacterial Indole
    • Authors: Shelby Steinmeyer; Daniel P Howsmon, Robert C. Alaniz, Juergen Hahn, Arul Jayaraman
      Abstract: Adoptive transfer of anti-inflammatory FOXP3+ Tregs has gained attention as a new therapeutic strategy for auto-inflammatory disorders such as Inflammatory Bowel Disease. The isolated cells are conditioned in vitro to obtain a sufficient number of anti-inflammatory FOXP3+ Tregs that can be reintroduced into the patient to potentially reduce the pathologic inflammatory response. Previous evidence suggests that microbiota metabolites can potentially condition cells during the in vitro expansion/differentiation step. However, the number of combinations of cytokines and metabolites that can be varied is large, preventing a purely experimental investigation which would determine optimal cell therapeutic outcomes. To address this problem, a combined experimental and modeling approached is investigated here: an artificial neural network model was trained to predict the steady-state T cell population phenotype after differentiation with a variety of host cytokines and the microbial metabolite indole. This artificial neural network model was able to both reliably predict the phenotype of these T cell populations and also uncover unexpected conditions for optimal Treg differentiation that were subsequently verified experimentally. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-01T07:40:24.730977-05:
      DOI: 10.1002/bit.26371
  • Enhanced isoprenoid production from xylose by engineered Saccharomyces
    • Authors: Suryang Kwak; Soo Rin Kim, Haiqing Xu, Guo-Chang Zhang, Stephan Lane, Heejin Kim, Yong-Su Jin
      Abstract: Saccharomyces cerevisiae has limited capabilities for producing fuels and chemicals derived from acetyl-CoA, such as isoprenoids, due to a rigid flux partition toward ethanol during glucose metabolism. Despite numerous efforts, xylose fermentation by engineered yeast harboring heterologous xylose metabolic pathways was not as efficient as glucose fermentation for producing ethanol. Therefore, we hypothesized that xylose metabolism by engineered yeast might be a better fit for producing non-ethanol metabolites. We indeed found that engineered S. cerevisiae on xylose showed higher expression levels of the enzymes involved in ethanol assimilation and cytosolic acetyl-CoA synthesis than on glucose. When genetic perturbations necessary for overproducing squalene and amorphadiene were introduced into engineered S. cerevisiae capable of fermenting xylose, we observed higher titers and yields of isoprenoids under xylose than glucose conditions. Specifically, co-overexpression of a truncated HMG1 (tHMG1) and ERG10 led to substantially higher squalene accumulation under xylose than glucose conditions. In contrast to glucose utilization producing massive amounts of ethanol regardless of aeration, xylose utilization allowed much less amounts of ethanol accumulation, indicating ethanol is simultaneously re-assimilated with xylose consumption and utilized for the biosynthesis of cytosolic acetyl-CoA. In addition, xylose utilization by engineered yeast with overexpression of tHMG1, ERG10, and ADS coding for amorphadiene synthase, and the down-regulation of ERG9 resulted in enhanced amorphadiene production as compared to glucose utilization. These results suggest that the problem of the rigid flux partition toward ethanol production in yeast during the production of isoprenoids and other acetyl-CoA derived chemicals can be bypassed by using xylose instead of glucose as a carbon source. This article is protected by copyright. All rights reserved
      PubDate: 2017-07-01T07:40:21.1953-05:00
      DOI: 10.1002/bit.26369
  • An adjustable multi-scale single beam acoustic tweezer based on ultrahigh
           frequency ultrasonic transducer
    • Authors: Xiaoyang Chen; Kwok–ho Lam, Ruimin Chen, Zeyu Chen, Ping Yu, Zhongping Chen, K. Kirk Shung, Qifa Zhou
      Abstract: This paper reports the fabrication, characterization and microparticle manipulation capability of an adjustable multi-scale single beam acoustic tweezer (SBAT) that is capable of flexibly changing the size of “tweezers” like ordinary metal tweezers with a single-element ultrahigh frequency (UHF) ultrasonic transducer. The measured resonant frequency of the developed transducer at 526 MHz is the highest frequency of piezoelectric single crystal based ultrasonic transducers ever reported. This focused UHF ultrasonic transducer exhibits a wide bandwidth (95.5% at −10 dB) due to high attenuation of high-frequency ultrasound wave, which allows the SBAT effectively excite with a wide range of excitation frequency from 150 MHz to 400 MHz by using the “piezoelectric actuator” model. Through controlling the excitation frequency, the wavelength of ultrasound emitted from the SBAT can be changed to selectively manipulate a single microparticle of different sizes (3–100 µm) by using only one transducer. This concept of flexibly changing “tweezers” size is firstly introduced into the study of SBAT. At the same time, it was found that this incident ultrasound wavelength play an important role in lateral trapping and manipulation for microparticle of different sizes. This article is protected by copyright. All rights reserved
      PubDate: 2017-06-27T07:39:11.815014-05:
      DOI: 10.1002/bit.26365
  • In vivo Synergistic Activity of a CAZyme Cassette from Acidothermus
           cellulolyticus Significantly Improves the Cellulolytic Activity of the C.
           bescii Exoproteome
    • Authors: Sun-Ki Kim; Daehwan Chung, Michael E. Himmel, Yannick J. Bomble, Janet Westpheling
      Abstract: The use of microbial cells to convert plant biomass directly to fuels and chemicals is referred to as consolidated bioprocessing (CBP). Members of the bacterial genus, Caldicellulosiruptor (Gram-positive, anaerobic hyperthermophiles) are capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This is accomplished by the production and secretion of free, multi-domain enzymes that outperform commercial enzyme cocktails on some substrates. Here, we show that the exoproteome of C. bescii may be enhanced by the heterologous expression of enzymes from Acidothermus cellulolyticus that act synergistically to improve sugar release from complex substrates; as well as improve cell growth. In this work, co-expression of the A. cellulolyticus Acel_0615 β-glucanase (GH6 and GH12) and E1 endoglucanase (GH5) enzymes resulted in an increase in the activity of the exoproteome on Avicel; as well as an increase in growth of C. bescii on Avicel compared to the parental strain or the strain expressing the β-glucanase alone. Our ability to engineer the composition and effectiveness of the exoproteome of these bacteria provides insight into the natural mechanism of plant cell wall deconstruction, as well as future directions for improving CBP. This article is protected by copyright. All rights reserved
      PubDate: 2017-06-26T05:32:31.2132-05:00
      DOI: 10.1002/bit.26366
  • Rational Engineering of p-Hydroxybenzoate Hydroxylase to Enable Efficient
           Gallic Acid Synthesis via A Novel Artificial Biosynthetic Pathway
    • Authors: Zhenya Chen; Xiaolin Shen, Jian Wang, Jia Wang, Qipeng Yuan, Yajun Yan
      Abstract: Gallic acid (GA) is a naturally occurring phytochemical that has strong antioxidant and antibacterial activities. It is also used as a potential platform chemical for the synthesis of diverse high-value compounds. Hydrolytic degradation of tannins by acids, bases or microorganisms serves as a major way for GA production, which however, might cause environmental pollution and low yield and efficiency. Here, we report a novel approach for efficient microbial production of GA. First, structure-based rational engineering of PobA, a p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa, generated a new mutant, Y385F/T294A PobA, which displayed much higher activity towards 3,4-dihydroxybenzoic acid (3,4-DHBA) than the wild type and any other reported mutants. Remarkably, expression of this mutant in Escherichia coli enabled generation of 1149.59 mg/L GA from 1000 mg/L 4-hydroxybenzoic acid (4-HBA), representing a 93% molar conversion ratio. Based on that, we designed and reconstituted a novel artificial biosynthetic pathway of GA and achieved 440.53 mg/L GA production from simple carbon sources in E. coli. Further enhancement of precursor supply through reinforcing shikimate pathway was able to improve GA de novo production to 1266.39 mg/L in shake flasks. Overall, this study not only led to the development of a highly active PobA variant for hydroxylating 3,4-DHBA into GA via structure-based protein engineering approach, but also demonstrated a promising pathway for bio-based manufacturing of GA and its derived compounds. This article is protected by copyright. All rights reserved
      PubDate: 2017-06-26T05:27:34.455351-05:
      DOI: 10.1002/bit.26364
  • Engineering CHO cells with an oncogenic KIT improves cells growth,
           resilience to stress and productivity
    • Authors: Mohamed Mahameed; Boaz Tirosh
      Abstract: An optimized biomanufacturing process in mammalian cells is contingent on the ability of the producing cells to reach high viable cell densities. In addition, at the peak of growth, cells need to continue producing the biological entity at a consistent quality. Thus, engineering cells with robust growth performance and resilience to variable stress conditions is highly desirable. The tyrosine kinase receptor, KIT, plays a key role in cell differentiation and the survival of several immune cell types. Its oncogenic mutant, D816V, endows cells with high proliferation capacity and resistance to kinase inhibitors. Importantly, this onco-KIT mutant when introduced into various cell types is arrested in the endoplasmic reticulum in a constitutively active form. Here, we investigated the effect of oncogenic D816V KIT on the performance of CHO-K1 cells under conventional tissue culture growth settings and when adapted, to shaking conditions. The onco-KIT promoted global protein synthesis, elevated the expression of a secretable transgene, enhanced proliferation, and improved the overall titers of a model glycoprotein. Moreover, the expression of the onco-KIT endowed the cells with a remarkable resistance to various stress conditions. Our data suggest that the introduction of onco-KIT can serve as a strategy for improving glycoprotein biomanufacturing. This article is protected by copyright. All rights reserved
      PubDate: 2017-06-19T07:06:10.792965-05:
      DOI: 10.1002/bit.26356
    • Authors: Pallab Datta; Veli Ozbolat, Bugra Ayan, Aman Dhawan, Ibrahim T. Ozbolat
      Abstract: Craniofacial (CF) tissue is an architecturally complex tissue consisting of both bone and soft tissues with significant patient specific variations. Conditions of congenital abnormalities, tumor resection surgeries, and traumatic injuries of the CF skeleton can result in major deficits of bone tissue. Despite advances in surgical reconstruction techniques, management of CF osseous deficits remains a challenge. Due its inherent versatility, bioprinting offers a promising solution to address these issues. In this review, we present and analyze the current state of bioprinting of bone tissue and highlight how these techniques may be adapted to serve regenerative therapies for CF applications. This article is protected by copyright. All rights reserved
      PubDate: 2017-06-10T07:25:20.915202-05:
      DOI: 10.1002/bit.26349
  • Biotechnology and Bioengineering: Volume 114, Number 11, November 2017
    • Pages: 2150 - 2154
      PubDate: 2017-09-26T11:09:53.105652-05:
      DOI: 10.1002/bit.26171
School of Mathematical and Computer Sciences
Heriot-Watt University
Edinburgh, EH14 4AS, UK
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Fax: +00 44 (0)131 4513327
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