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Biotechnology and Bioengineering

Dario Vazquez-Albacete, Ana Mafalda Cavaleiro, Ulla Christensen, Susanna Seppälä, Birger Lindberg Møller, Morten H H Nørholm
Membrane-associated Cytochromes P450 (P450s) are one of the most important enzyme families for biosynthesis of plant-derived medicinal compounds. However, the hydrophobic nature of P450s makes their use in robust cell factories a challenge. Here we explore a small library of N-terminal expression tag chimeras of the model plant P450 CYP79A1 in different Escherichia coli strains. Using a high-throughput screening platform based on C-terminal GFP fusions, we identify several highly expressing and robustly performing chimeric designs...
October 17, 2016: Biotechnology and Bioengineering
Carolyn N Haarmeyer, Matthew D Smith, Shishir Chundawat, Deanne Sammond, Timothy A Whitehead
Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue towards energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption...
October 17, 2016: Biotechnology and Bioengineering
Benjamin Kick, Samantha Hensler, Florian Praetorius, Hendrik Dietz, Dirk Weuster-Botz
The bacteriophage M13 has found frequent applications in nanobiotechnology due to its chemically and genetically tunable protein surface and its ability to self-assemble into colloidal membranes. Additionally, its single-stranded (ss) genome is commonly used as scaffold for DNA origami. Despite the manifold uses of M13, upstream production methods for phage and scaffold ssDNA are underexamined with respect to future industrial usage. Here, the high-cell-density phage production with Escherichia coli as host organism was studied in respect of medium composition, infection time, multiplicity of infection and specific growth rate...
October 17, 2016: Biotechnology and Bioengineering
Ines Westphal, Claudia Jedelhauser, Gregor Liebsch, Arnd Wilhelmi, Attila Aszodi, Matthias Schieker
Bone tissue engineering (BTE) utilizing biomaterial scaffolds and human mesenchymal stem cells (hMSCs) is a promising approach for the treatment of bone defects. The quality of engineered tissue is crucially affected by numerous parameters including cell density and the oxygen supply. In this study, a novel oxygen-imaging sensor was introduced to monitor the oxygen distribution in three dimensional (3D) scaffolds in order to analyze a new cell-seeding strategy. Immortalized hMSCs, pre-cultured in a monolayer for 30-40% or 70-80% confluence, were used to seed demineralized bone matrix (DBM) scaffolds...
October 17, 2016: Biotechnology and Bioengineering
Ward Blanken, Stefan Schaap, Sophie Theobald, Arjen Rinzema, René H Wijffels, Marcel Janssen
The loss of carbon dioxide (CO2 ) to the environment during microalgae cultivation is undesirable for both environmental and economic reasons. In this study, a phototrophic biofilm growth model was developed and validated with the objective to maximize both CO2 utilization efficiency and production of microalgae in biofilms. The model was validated in growth experiments with CO2 as the limiting substrate. The CO2 utilization and biomass productivity were maximized by changing the gas flow rate, the number of biofilm reactors in series, and gas composition...
October 17, 2016: Biotechnology and Bioengineering
Evangelia Hondroulis, Pooja Sabhachandani, Alexandru Movila, Saheli Sarkar, Noa Cohen, Toshihisa Kawai, Tania Konry
Microfluidic droplets are used to isolate cell pairs and prevent crosstalk with neighboring cells while permitting free motility and interaction within the confined space. Dynamic analysis of cellular heterogeneity in droplets has provided insights in various biological processes. Droplet manipulation methods such as fusion and fission make it possible to precisely regulate the localized environment of a cell in a droplet and deliver reagents as required. Droplet fusion strategies achieved by passive mechanisms preserve cell viability and are easier to fabricate and operate...
October 10, 2016: Biotechnology and Bioengineering
Jian-Feng Huang, Zhi-Qiang Liu, Li-Qun Jin, Xiao-Ling Tang, Zhen-Yang Shen, Huan-Huan Yin, Yu-Guo Zheng
L-methionine has attracted a great deal of attention for its nutritional, pharmaceutical, and clinical applications. In this study, Escherichia coli W3110 was engineered via deletion of a negative transcriptional regulator MetJ and over-expression of homoserine O-succinyltransferase MetA together with efflux transporter YjeH, resulting in L-methionine overproduction which is up to 413.16 mg/L. The partial inactivation of the L-methionine import system MetD via disruption of metI made the engineered E. coli ΔmetJ ΔmetI/pTrcA*H more tolerant to high L-ethionine concentration and accumulated L-methionine to a level 43...
October 10, 2016: Biotechnology and Bioengineering
Song Jiao, Xu Li, Huimin Yu, Huan Yang, Xue Li, Zhongyao Shen
Surfactin-family lipopeptides are green biosurfactants with substantial industrial potential. The major problem prohibiting surfactin use is the low titer of the wild producer, Bacillus subtilis. Using transcriptomic analysis, four strong promoters, PgroE, Pcdd, PrplK, and PsspE, were identified and cloned from the genome of B. subtilis THY-7, a novel surfactin producer that has been identified from soil with a 0.55 g/L surfactin titer. An optimal promoter, PgroE, was selected to replace the native THY-7 surfactin synthase (SrfA) promoter through single-cross homologous recombination; however, the resulting engineered strain containing the PgroE substitution did not synthesize surfactin...
October 10, 2016: Biotechnology and Bioengineering
Zhen Chen, Peize Liu, Zhipeng Li, Wencheng Yu, Zhi Wang, Haosheng Yao, Yuanpeng Wang, Qingbiao Li, Xu Deng, Ning He
The present study reports the sequenced genome of Bacillus licheniformis CGMCC 2876, which is composed of a 4,284,461 bp chromosome that contains 4,188 protein-coding genes, 72 tRNA genes, and 21 rRNA genes. Additional analysis revealed an eps gene cluster with 16 open reading frames. Conserved Domains Database analysis combined with qPCR experiments indicated that all genes in this cluster were involved in polysaccharide bioflocculant synthesis. Phosphoglucomutase and UDP-glucose pyrophosphorylase were supposed to be key enzymes in polysaccharide secretion in B...
October 6, 2016: Biotechnology and Bioengineering
Marta Ondrésik, Fatima R Azevedo Maia, Alain da Silva Morais, Ana C Gertrudes, Ana H Dias Bacelar, Cristina Correia, Cristiana Gonçalves, Hajer Radhouani, Rui Amandi Sousa, Joaquim M Oliveira, Rui L Reis
Osteoarthritis (OA) affects a large number of the population, and its incidence is showing a growing trend with the increasing life span. OA is the most prevalent joint condition worldwide, and currently, there is no functional cure for it. This review seeks to briefly overview the management of knee OA concerning standardized pharmaceutical and clinical approaches, as well as the new biotechnological horizons of OA treatment. The potential of biomaterials and state of the art of advanced therapeutic approaches, such as cell and gene therapy focused primarily on cartilage regeneration are the main subjects of this review...
October 5, 2016: Biotechnology and Bioengineering
Renliang Huang, Hong Guo, Rongxin Su, Wei Qi, Zhimin He
Recycling cellulases by substrate adsorption is a promising strategy for reducing the enzyme cost of cellulosic ethanol production. However, β-glucosidase has no carbohydrate-binding module (CBM). Thus, additional enzymes are required in each cycle to achieve a high ethanol yield. In this study, we report a new method of recycling cellulases without β-glucosidase supplementation using lignocellulosic substrate, an engineered strain expressing β-glucosidase and Tween 80. The cellulases and Tween 80 were added to an aqueous suspension of diluted sulfuric acid/ammonia-treated corncobs in a simultaneous saccharification and fermentation (SSF) process for ethanol production...
October 3, 2016: Biotechnology and Bioengineering
Hongzhong Lu, Weiqiang Cao, Liming Ouyang, Jianye Xia, Mingzhi Huang, Ju Chu, Yingping Zhuang, Siliang Zhang, Henk Noorman
Aspergillus niger is one of the most important cell factories for industrial enzymes and organic acids production. A comprehensive genome-scale metabolic network model (GSMM) with high quality is crucial for efficient strain improvement and process optimization. The lack of accurate reaction equations and gene-protein-reaction associations (GPRs) in the current best model of A. niger named GSMM iMA871, however, limits its application scope. To overcome these limitations, we updated the A. niger GSMM by combining the latest genome annotation and literature mining technology...
October 3, 2016: Biotechnology and Bioengineering
M N Cruz Bournazou, T Barz, D Nickel, D Lopez Cárdenas, F Glauche, A Knepper, P Neubauer
We present an integrated framework for the online optimal experimental re-design of parallel nonlinear dynamic processes that aims to precisely estimate the parameter set of macro kinetic growth models with minimal experimental effort. This provides a systematic solution for rapid validation of a specific model to new strains, mutants, or products. In biosciences, this is especially important as model identification is a long and laborious process which is continuing to limit the use of mathematical modeling in this field...
October 3, 2016: Biotechnology and Bioengineering
Nardrapee Karuna, Tina Jeoh
Cellulosic biomass is the most promising feedstock for renewable biofuel production; however, the mechanisms of the heterogeneous cellulose saccharification reaction are still unsolved. As cellulases need to bind isolated molecules of cellulose at the surface of insoluble cellulose fibrils or larger aggregated cellulose structures in order to hydrolyze glycosidic bonds, the "accessibility of cellulose to cellulases" is considered to be a reaction limiting property of cellulose. We have defined the accessibility of cellulose to cellulases as the productive binding capacity of cellulose, that is, the concentration of productive binding sites on cellulose that are accessible for binding and hydrolysis by cellulases...
October 3, 2016: Biotechnology and Bioengineering
Søren Heintz, Tim Börner, Rolf H Ringborg, Gustav Rehn, Carl Grey, Mathias Nordblad, Ulrich Krühne, Krist V Gernaey, Patrick Adlercreutz, John M Woodley
An experimental platform based on scaled-down unit operations combined in a plug-and-play manner enables easy and highly flexible testing of advanced biocatalytic process options such as in situ product removal (ISPR) process strategies. In such a platform, it is possible to compartmentalize different process steps while operating it as a combined system, giving the possibility to test and characterize the performance of novel process concepts and biocatalysts with minimal influence of inhibitory products. Here the capabilities of performing process development by applying scaled-down unit operations are highlighted through a case study investigating the asymmetric synthesis of 1-methyl-3-phenylpropylamine (MPPA) using ω-transaminase, an enzyme in the sub-family of amino transferases (ATAs)...
September 26, 2016: Biotechnology and Bioengineering
Xiaoxiang Dong, Xiulai Chen, Yuanyuan Qian, Yuancai Wang, Li Wang, Weihua Qiao, Liming Liu
A four-carbon dicarboxylic acid L-malate has recently attracted attention due to its potential applications in the fields of medicine and agriculture. In this study, Escherichia coli W3110 was engineered and optimized for L-malate production via one-step L-malate synthesis pathway. First, deletion of the genes encoding lactate dehydrogenase (ldhA), pyruvate oxidase (poxB), pyruvate formate lyase (pflB), phosphotransacetylase (pta), and acetate kinase A (ackA) in pta-ackA pathway led to accumulate 20.9 g/L pyruvate...
September 26, 2016: Biotechnology and Bioengineering
Salina Louie, Benjamin Haley, Brett Marshall, Amy Heidersbach, Mandy Yim, Martina Brozynski, Danming Tang, Cynthia Lam, Bronislawa Petryniak, David Shaw, Jeongsup Shim, Aaron Miller, John B Lowe, Brad Snedecor, Shahram Misaghi
During antibody dependent cell cytotoxicity (ADCC) the target cells are killed by monocytes and natural killer cells. ADCC is enhanced when the antibody heavy chain's core N-linked glycan lacks the fucose molecule(s). Several strategies have been utilized to generate fully afucosylated antibodies. A commonly used and efficient approach has been knocking out the FUT8 gene of the Chinese hamster ovary (CHO) host cells, which results in expression of antibody molecules with fully afucosylated glycans. However, a major drawback of the FUT8-KO host is the requirement for undertaking two separate cell line development (CLD) efforts in order to obtain both primarily fucosylated and fully afucosylated antibody species for comparative studies in vitro and in vivo...
September 26, 2016: Biotechnology and Bioengineering
Lisa Mears, Stuart M Stocks, Mads O Albaek, Gürkan Sin, Krist V Gernaey
A mechanistic model-based soft sensor is developed and validated for 550L filamentous fungus fermentations operated at Novozymes A/S. The soft sensor is comprised of a parameter estimation block based on a stoichiometric balance, coupled to a dynamic process model. The on-line parameter estimation block models the changing rates of formation of product, biomass, and water, and the rate of consumption of feed using standard, available on-line measurements. This parameter estimation block, is coupled to a mechanistic process model, which solves the current states of biomass, product, substrate, dissolved oxygen and mass, as well as other process parameters including kL a, viscosity and partial pressure of CO2 ...
September 19, 2016: Biotechnology and Bioengineering
Joaquina X Mascarenhas, Nikolay Korokhov, Lisa Burger, Ademola Kassim, Jason Tuter, Daniel Miller, Trissa Borgschulte, Henry J George, Audrey Chang, David J Pintel, David Onions, Kevin J Kayser
Contamination by the parvovirus minute virus of mice (MVM) remains a challenge in Chinese hamster ovary (CHO) biopharmaceutical production processes. Although infrequent, infection of a bioreactor can be catastrophic for a manufacturer, can impact patient drug supply and safety, and can have regulatory implications. We evaluated engineering a CHO parental cell line (CHOZN(®) GS(-/-) ) to create a new host cell line that is resistant to MVM infection by modifying the major receptors used by the virus to enter cells...
September 19, 2016: Biotechnology and Bioengineering
Ali Halalipour, Michael R Duff, Elizabeth E Howell, José I Reyes-De-Corcuera
High hydrostatic pressure (HHP) stabilized glucose oxidase (GOx) against thermal inactivation. The apparent first-order kinetics of inactivation of GOx were investigated at 0.1-300 MPa and 58.8-80.0 °C. At 240 MPa and 74.5 °C, GOx inactivated at a rate 50 times slower than at atmospheric pressure at the same temperature. The apparent activation energy of inactivation at 300 MPa was 281.0 ± 17.4 kJ mol(-1) or 1.3 fold smaller than for the inactivation at atmospheric pressure (378.1 ± 25...
September 19, 2016: Biotechnology and Bioengineering
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