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Tuesday, 26 September 2017
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Biotechnology and Bioengineering
Wiley Online Library : Biotechnology and Bioengineering

  • Engineering of cell membrane to enhance heterologous production of hyaluronic acid in Bacillus subtilis
    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

  • Intracellular Response to Process Optimization and Impact on Productivity and Product Aggregates for a high?titer CHO Cell Process
    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

  • Advancements in the design and scalable production of viral gene transfer vectors
    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

  • Burkholderia cepacia lipase: A versatile catalyst in synthesis reactions
    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

  • Reconstruction of Lignin and Hemicelluloses by Aqueous Ethanol Anti?solvents to Improve the Ionic Liquid?Acid Pretreatment Performance of Arundo donax Linn
    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

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