Virus-Induced Gene Silencing (VIGS) is a rapid technique that allows for specific and reproducible post transcriptional degradation of targeted mRNA. The method has been proven efficient for suppression of expression of many single enzymes. The metabolic networks of plants, however, often contain isoenzymes and gene families which are able to compensate for a mutation and mask the development of a silencing phenotype. Here we show the application of multiple gene VIGS repression for the study of these redundant biological pathways. Several genes in the starch degradation pathway (DPE1, DPE2 and GWD) were silenced. The functionally distinct DPE enzymes are present in alternate routes for sugar export to the cytoplasm and result in an increase in starch when silenced individually. Simultaneous silencing of DPE1 and DPE2 in Nicotiana benthamiana resulted in a near complete suppression in starch and accumulation of malto-oligosaccharides.

The Gateway Technology cloning system and the transposon technology represent state-of-the-art laboratory techniques. Combination of these molecular tools may allow rapid cloning of target genes into expression vectors. Here, we describe a novel Gateway Technology compatible transposon plasmid that combines the advantages of the Gateway recombination cloning with the Sleeping Beauty (SB) transposon-mediated transgene integrations. In our system the transposition is catalyzed by the novel hyperactive SB100x transposase providing the high efficient and precise transgene integrations into the host genome. A Gateway compatible transposon plasmid was generated in which the potential target gene may be fused with a Yellow Fluorescent Protein (YFP) tag at the N'- terminal. The vector utilizes the CAGGS promoter to control the fusion protein expression. The transposon expression vector encoding fusion YFP-IFNB1 protein together with the hyperactive SB100x transposase was used to generate stable cell lines in human embryonic kidney (HEK293) and rat adipose-derived stromal cells (ASC). HEK293 and ASCs stably expressed and secreted human interferon-beta protein up to 4 weeks after transfection. The generated Gateway compatible transposon plasmid may be utilized for numerous experimental approaches, such as gene therapy or high-throughput screening methods in primary cells, representing a valuable molecular tool for laboratory applications.

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is industrially produced by fermentation using Saccharomyces cerevisiae. Before the glutathione fermentation process with S. cerevisiae, a glucose extraction process from starchy materials is required. This glucose extraction is usually carried out by converting starchy materials to starch using high-temperature cooking and subsequent hydrolysis by amylases to convert starch to glucose. In this study, to develop an energy-saving glutathione production process by reducing energy consumption during the cooking step, we efficiently produced glutathione from low-temperature cooked rice using amylase-expressing S. cerevisiae. The combination of the amylase-expressing yeast with low-temperature cooking is potentially applicable to a variety of energy-saving bio-production methods of chemicals from starchy bio-resources.

Pyruvate kinase-deficient Escherichia coli (PB25) is a low by-product producing yet fast growing strain that has been shown to have technological potential. Flux bounding through finding the extreme point flux sets was previously reported to identify highly variable fluxes and thus alternate metabolite trafficking scenarios. Previously, the extreme point flux sets were used to design tracer experiments, however, variation in extracellular measurements was not considered, and reaction reversibility was assumed to be low to moderate. In this study, we examined further the utility of bounding the fluxes and predetermining the trafficking scenarios in the PB25 problem, including confirmation of quasi-linearity between extreme points to ensure sensitivity is maintained. The effects of variation in extracellular measurements and reaction reversibilities were also examined. Tightened flux bounds reduced the nonlinearity between label distribution and fluxes. For low to moderate reversibility, contrast was also preserved. However, for highly reversible phosphoglucoisomerase activity, information from common analytes could lead to a flux solution that is biased towards one extreme point. Based on the PB25 model, some suggestions are provided for how pre-determining flux bounds and trafficking scenarios could enable flux identification in larger network problems.

Commercial-scale cellulosic ethanol production has been hindered by high costs associated with cellulose-to-glucose conversion and hexose and pentose co-fermentation. Simultaneous saccharification and fermentation (SSF) with a yeast strain capable of xylose and cellobiose co-utilization has been proposed as a possible avenue to reduce these costs. The recently developed DA24-16 strain of S. cerevisiae incorporates a xylose assimilation pathway and a cellodextrin transporter (CDT) that permit rapid growth on xylose and cellobiose. In the present work, a mechanistic kinetic model of cellulase-catalyzed hydrolysis of cellulose was combined with a multi-substrate model of microbial growth to investigate the ability of DA24-16 and improved cellobiose-consuming strains to obviate the need for exogenously added β-glucosidase and to assess the impact of cellobiose utilization on SSF and separate hydrolysis and fermentation (SHF). Results indicate that improved CDT-containing strains capable of growing on cellobiose as rapidly as on glucose produced ethanol nearly as rapidly as non-CDT-containing yeast supplemented with β-glucosidase. In producing 75 g/L ethanol, SSF with any strain did not result in shorter residence times than SHF with a 12-hour saccharification step. Strains with improved cellobiose utilization are therefore unlikely to allow higher titers to be reached more quickly in SSF than in SHF.

The folding of many cellular proteins occurs co-translationally immediately outside the ribosome exit tunnel, where ribosomal proteins and other associated factors coordinate the synthesis and folding of newly translated polypeptides. Here, we show that the large subunit protein L29, which forms part of the exit tunnel in Escherichia coli, is required for the productive synthesis of an array of structurally diverse recombinant proteins including the green fluorescent protein (GFP) and an intracellular single-chain Fv antibody. Surprisingly, the corresponding mRNA transcript level of these proteins was markedly less abundant in cells lacking L29, suggesting an unexpected regulatory mechanism that links defects in the exit tunnel to the expression of genetic information. To further highlight the importance of L29 in maintaining protein expression, we used rounds of mutagenesis and selection to obtain L29 variants that enhanced GFP expression. Overall, our results suggest that the ribosomal exit tunnel proteins may be key targets for optimizing the overproduction of active, structurally complex recombinant proteins in bacterial cells.

Alcohol-based liquid fuels feature significantly in the political and social agendas of many countries, seeking energy sustainability. It is certain that ethanol will be the entry point for many sustainable processes. Conventional ethanol production using maize- and sugarcane-based carbohydrates with Saccharomyces cerevisiae is well established, while lignocellulose-based processes are receiving growing interest despite posing greater technical and scientific challenges. A significant challenge that arises from the chemical hydrolysis of lignocellulose is the generation of toxic compounds in parallel with the release of sugars. These compounds, collectively termed pre-treatment inhibitors, impair metabolic functionality and growth. Their removal, pre-fermentation or their abatement, via milder hydrolysis, are currently uneconomic options. It is widely acknowledged that a more cost effective strategy is to develop resistant process strains. Here we describe and classify common inhibitors and describe in detail the reported physiological responses that occur in second-generation strains, which include engineered yeast and mesophilic and thermophilic prokaryotes. It is suggested that a thorough understanding of tolerance to common pre-treatment inhibitors should be a major focus in ongoing strain engineering. This review is a useful resource for future metabolic engineering strategies.

Understanding physiological responses to fermentation inhibitors is crucial to the delivery of a successful bioprocess. Pre-treatment methods inevitably generate a number of biologically detrimental compounds alongside the carbohydrates that are liberated from biomass. Inhibitors impact efficiency through decreasing both biomass and product yields. A thorough understanding of tolerance to pre-treatment inhibitors should be a major focus in ongoing strain engineering. This review can be used as a foundation that guides future metabolic engineering strategies.

Extracellular fungal flavocytochrome cellobiose dehydrogenase (CDH) is a promising enzyme for both bioelectronics and lignocellulose bioconversion. A selective high-throughput screening assay for CDH in the presence of various fungal oxidoreductases was developed. It is based on Prussian Blue (PB) in situ formation in the presence of cellobiose (<0.25 mM), ferric acetate, and ferricyanide. CDH induces PB formation via both reduction of ferricyanide to ferrocyanide reacting with an excess of Fe³⁺ (pathway 1) and reduction of ferric ions to Fe²⁺ reacting with the excess of ferricyanide (pathway 2). Basidiomycetous and ascomycetous CDH formed PB optimally at pH 3.5 and 4.5, respectively. In contrast to the holoenzyme CDH, its FAD-containing dehydrogenase domain lacking the cytochrome domain formed PB only via pathway 1 and was less active than the parent enzyme. The assay can be applied on active growing cultures on agar plates or on fungal culture supernatants in 96-well plates under aerobic conditions. Neither other carbohydrate oxidoreductases (pyranose dehydrogenase, FAD-dependent glucose dehydrogenase, glucose oxidase) nor laccase interfered with CDH activity in this assay. Applicability of the developed assay for the selection of new ascomycetous CDH producers as well as possibility of the controlled synthesis of new PB nanocomposites by CDH are discussed.

Cellobiose dehydrogenases (CDHs) are extracellular fungal flavocytochromes which, owing to their unique electron transfer properties, are extensively studied in bioelectronics. Neutral CDHs from ascomycetes are of particular interest because of their possible application in the physiologically compatible nanobiodevices. In this article, the authors present a new selective and high-throughput screening assay for these enzymes based on in situ Prussian Blue formation.

Proteolysis during fermentation may have a severe impact on the yield and quality of a secreted product. In the current study, we demonstrate the use of high-gradient magnetic fishing (HGMF) as an efficient alternative to the more conventional methods of preventing proteolytic degradation. Bacitracin-linked magnetic affinity adsorbents were employed directly in a fermenter during Bacillus licheniformis cultivation to remove trace amounts of unwanted proteases. The constructed magnetic adsorbents had excellent, highly specific binding characteristics in the fermentation broth (K_d = 1.94 micromolar; Q_max = 222.8 mg/g), which obeyed the Langmuir isotherm and had rapid binding kinetics (equilibrium in <300 s). When applied directly in shake-flask cultures or in a 1-L fermenter and then removed by HGMF, the degradation of the model protein bovine serum albumin was stopped. The adsorbents could be recycled and reused during the same fermentation to remove freshly produced proteases, extending the life of the model protein in the fermenter. HGMF may provide an efficient method of stabilizing heterologous proteins produced in cultivation processes.

Degradation or modification of products during fermentation and downstream processing complicates both laboratory and industrial scale production, which is often a side-effect extracellular enzyme by-products. In this article, authors from the Technical University of Denmark, demonstrate the use of high-gradient magnetic fishing (HGMF) as an efficient alternative to the more conventional methods of preventing proteolytic degradation.

Rayleigh-Bénard convective PCR is a simple and effective design for amplification of DNA. Convective PCR is, however, extremely sensitive to environmental temperature fluctuations, especially when using small- diameter test tubes. Therefore, this method is inherently unstable with limited applications. Here, we present a convective PCR device that has been modified by adding thermal baffles. With this thermally baffled device the influence from fluctuations in environmental temperature were significantly reduced, even in a wind tunnel (1 m/s). The thermally baffled PCR instrument described here has the potential to be used as a low-cost, point-of-care device for PCR-based molecular diagnostics in the field.

Thermally baffled device can reduce the environmental influence on convective PCR amplification: Convective PCR is extremely sensitive to environmental temperature fluctuations, and is inherently unstable with limited applications. In this study the authors have designed a thermally baffled convective PCR device with thermal baffles, which significantly reduces the influence from environmental temperature fluctuations, while PCR amplification is carried out successfully in a short reaction time. With its simple design the thermally baffled PCR instrument can provide a powerful tool for point-of-care molecular diagnostics in the field.

Many recent advances in bioprocessing have been enabled by developments in miniaturization and microfluidics. A continuing challenge, however, is integrating multiple unit operations that require distinct spatial boundaries, especially with included labile biological components. We have suggested “biofabrication” as a means for organizing cells and biomolecules in complex configurations while preserving function of individual components. Polysaccharide films of chitosan and alginate that are assembled on-chip by electrodeposition are “smart” configurable interfaces that mediate communication between the biological systems and microfabricated devices. Here, we demonstrate the scalable performance of a production address, where incubated cells secrete antibodies, and a capture address, where secreted antibody is retained with specificity and subsequently assayed. The antibody exchange from one electro-address to another exemplifies integrated in-film bioprocessing, facilitated by the integrated biofabrication techniques used. This in-film approach enables complex processes without need for microfluidics and valving. Finally, we have shown scalability by reducing electrode sizes to a 1 mm scale without compromising film biofabrication or bioprocessing performance. The in situ reversible deposition of viable cells, productivity characterization, and capture of secreted antibodies could find use in bioprocessing applications such as clonal selection, run-to-run monitoring, initial scale-up, and areas including drug screening and biopsy analysis.

Many recent advances in bioprocessing have been enabled by developments in miniaturization and microfluidics. A continuing challenge, however, is integrating multiple unit operations that require distinct spatial boundaries, especially with included labile biological components. In this article, the authors demonstrate “in-film” bioprocessing, which has diverse application potential such as in drug screening and biopsy analysis.

Advances in fundamental physical and optical principles applied to novel fluorescence methods are currently resulting in rapid progress in cell biology and physiology. Instrumentation devised in pioneering laboratories is becoming commercially available, and study findings are now becoming accessible. The first results have concerned mainly higher eukaryotic cells but many more developments can be expected, especially in microbiology. Until now, some important problems of cell physiology have been difficult to investigate due to interactions between probes and cells, excretion of probes from cells and the inability to make in situ observations deep within the cell, within tissues and structures. These technologies will enable microbiologists to address these topics. This Review aims at introducing the limits of current physiology evaluation techniques, the principles of new fluorescence technologies and examples of their use in this field of research for evaluating the physiological state of cells in model media, biofilms or tissue environments. Perspectives on new imaging technologies, such as super-resolution imaging and non-linear highly sensitive Raman microscopy, are also discussed. This review also serves as a reference to those wishing to explore how fluorescence technologies can be used to understand basic cell physiology in microbial systems.

Advances in fundamental physical and optical principles and their application in microscopy have enabled us to make rapid progress in cell biology and physiology. This review comprehensively evaluates the limits of current physiology evaluation techniques and principles of new fluorescent technologies, and serves as an excellent guide to those wishing to explore how fluorescence technologies can be used to understand basic cell physiology in microbial systems.

See accompanying article by Pham et al. DOI: 10.1002/biot.201100430

Production of recombinant proteins in plant cell or organ cultures and their secretion into the plant cell culture medium simplify the purification procedure and increase protein yield. In this study, the sweet-tasting protein thaumatin I was expressed and successfully secreted from tobacco hairy root cultures. The presence of an ER signal peptide appears to be crucial for the secretion of thaumatin: without an ER signal peptide, no thaumatin was detectable in the spent medium, whereas inclusion of the ER signal peptide calreticulin fused to the N terminus of thaumatin led to the secretion of thaumatin into the spent medium of hairy root cultures at concentrations of up to 0.21 mg/L. Extracellular thaumatin levels reached a maximum after 30 days (stationary phase) and the subsequent decline was linked to the rapid increase of proteases in the medium. Significant amounts of thaumatin were trapped in the apoplastic space of the root cells. The addition of polyvinylpyrrolidone and sodium chloride into the culture medium led to an increase of extracellular thaumatin amounts up to 1.4 and 2.63 mg/L, respectively. Thaumatin production compares well with yields from other transgenic plants, so that tobacco hairy roots can be considered an alternative production platform of thaumatin.

See accompanying commentary by Eva Stoger DOI: 10.1002/biot.201100472

Rhizosecretion of recombinant thaumatin: Tobacco hairy root cultures are a suitable expression platform for the production and secretion of recombinant thaumatin. Thaumatin is a sweet-tasting protein with high potential to serve as a substitute for sugars or commonly used sweeteners.

Acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum has been extensively studied in recent years because the organism is recognized as an excellent butanol producer. A parallel bioreactor system with 48 stirred-tank bioreactors on a 12 mL scale was evaluated for batch cultivations of the strictly anaerobic, butanol-producing C. acetobutylicum ATCC 824. Continuous gassing with nitrogen gas was applied to control anaerobic conditions. Process performances of ABE batch fermentations on a milliliter scale were identical to the liter-scale stirred-tank reactor if reaction conditions were identical on the different scales (e.g., initial medium, pH, temperature, specific evaporation rates, specific power input by the stirrers). The effects of varying initial ammonia concentrations (0.1–4.4 g L⁻¹) were studied in parallel with respect to glucose consumption and butanol production of C. acetobutylicum ATCC 824 as a first application example. The highest butanol yield of 33% (mol mol⁻¹) was observed at initial ammonia concentrations of 0.5 and 1.1 g L⁻¹. This is the first report on the successful application of a 48 parallel stirred-tank bioreactor system for reaction engineering studies of strictly anaerobic microorganisms at the milliliter scale.

Application of an anaerobic stirred-tank screening system for reaction engineering studies on biofuel production: Continuous gassing with nitrogen gas was applied for the control of anaerobic conditions in 48 parallel stirred-tank bioreactors on a mL-scale. Scalability to the liter-scale was shown with batch cultivations of a strictly anaerobic butanol-producing Clostridium acetobutylicum. The anaerobic parallel stirred-tank bioreactors can thus be applied as a scalable screening system for reaction engineering studies of varying recombinant producer strains at varying process conditions.

Fed batch culture processes are often characterized by decreasing cell culture performance as the process continues, presumably through the depletion of vital nutrients and the accumulation of toxic byproducts. We have similarly observed that cellular productivity (Qp) often declines during the course of a fed batch process; however, it is not clear why some cell lines elicit this behavior, while others do not. We here present a transcriptomic profiling analysis of a phenotype of sustained Qp (S-Qp) in production Chinese hamster ovary (CHO) culture, in which a marked drop in Qp levels (“non-sustained” (NS) phenotype) in two cell lines irrespective of viability levels was compared to two cell lines that consistently displayed high Qp throughout the culture (“sustained” (S) phenotype). Statistical analysis of the microarray data resulted in the identification of 22 gene transcripts whose expression patterns were either significantly negatively or positively correlated with long-term maintenance of Qp over the culture lifespan. qPCR analysis of four of these genes on one of each (NS2, S2) of the cell lines examined by microarray analysis confirmed that two genes (CRYAB and MGST1) both replicated the microarray results and were differentially regulated between the NS and S phenotypes.

Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility-enhancing and affinity tags. Furthermore, we provide summaries of well-characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology. This review serves as an excellent literature reference for those working on protein fusion tags.

Protein fusion tags are indispensible tools in protein expression and purification studies: This review highlights various protein fusion tags, including the calmodulin-binding peptide, and their use for protein isolation and purification, including best practices for fusion constructs. This information should be a useful reference to a broad audience, from experts to those new to the protein expression field.

Mathematical models have substantially improved our ability to predict the response of a complex biological system to perturbation, but their use is typically limited by difficulties in specifying model topology and parameter values. Additionally, incorporating entities across different biological scales ranging from molecular to organismal in the same model is not trivial. Here, we present a framework called “querying quantitative logic models” (Q2LM) for building and asking questions of constrained fuzzy logic (cFL) models. cFL is a recently developed modeling formalism that uses logic gates to describe influences among entities, with transfer functions to describe quantitative dependencies. Q2LM does not rely on dedicated data to train the parameters of the transfer functions, and it permits straight-forward incorporation of entities at multiple biological scales. The Q2LM framework can be employed to ask questions such as: Which therapeutic perturbations accomplish a designated goal, and under what environmental conditions will these perturbations be effective? We demonstrate the utility of this framework for generating testable hypotheses in two examples: (i) a intracellular signaling network model; and (ii) a model for pharmacokinetics and pharmacodynamics of cell-cytokine interactions; in the latter, we validate hypotheses concerning molecular design of granulocyte colony stimulating factor.

Querying Quantitative Logic Models (Q2LM) to study intracellular signaling networks and cell/cytokine interactions: The authors present a framework for building and asking questions of constrained fuzzy logic (cFL) models constructed based on prior knowledge. We demonstrate the utility of this framework for generating testable hypotheses in an intracellular signaling network model and a model for pharmacokinetics and pharmacodynamics of G-CSF.

Variability in product accumulation is one of the major obstacles limiting the widespread commercialization of plant cell culture technology to supply natural product pharmaceuticals. Despite extensive process engineering efforts, which have led to increased yields, plant cells exhibit variability in productivity that is poorly understood. Elicitation of Taxus cultures with methyl jasmonate (MeJA) induces paclitaxel accumulation, but to varying extents in different cultures. In the current study, cultures with different aggregation profiles were established to create predictable differences in paclitaxel accumulation upon MeJA elicitation. Expression of known paclitaxel biosynthetic genes in MeJA-elicited cultures exhibiting both substantial (15-fold) and moderate (2-fold) differences in paclitaxel accumulation was analyzed using quantitative reverse transcriptase PCR. Each population exhibited the characteristic large increase in paclitaxel pathway gene expression following MeJA elicitation; however, differences in expression between populations were minor, and only observed for the cultures with the 15-fold variation in paclitaxel content. These data suggest that although upregulation of biosynthetic pathway gene expression contributes to observed increases in paclitaxel synthesis upon elicitation with MeJA, there are additional factors that need to be uncovered before paclitaxel productivity can be fully optimized.

Paclitaxel is a chemotherapeutic agent used for the treatment of several cancer types. Although plant cells cultures have variable productivity, it has nevertheless become a sustainable source of paclitaxel supply. In this article, the authors correlate gene expression levels to accumulation of the compound – it is the first study to compare gene expression in cultures with variable levels of paclitaxel accumulation, providing valuable information regarding the regulation of paclitaxel synthesis.

In vitro cell culture models of the blood–brain barrier (BBB) are important tools used to study cellular physiology and brain disease therapeutics. Although the number of model configurations is expanding across neuroscience laboratories, it is not clear that any have been effectively optimized. A sequential screening study to identify optimal primary mouse endothelial cell parameter set points, grown alone and in combination with common model enhancements, including co-culturing with primary mouse or rat astrocytes and addition of biochemical agents in the media, was performed. A range of endothelial cell-seeding densities (1–8 × 10⁵ cells/cm²) and astrocyte-seeding densities (2–8 × 10⁴ cells/cm²) were studied over seven days in the system, and three distinct media-feeding strategies were compared to optimize biochemical agent exposure time. Implementation of all optimal set points increased transendothelial electrical resistance by over 200% compared to an initial model and established a suitable in vitro model for brain disease application studies. These results demonstrate the importance of optimizing cell culture growth, which is the most important parameter in creating an in vitro BBB model as it directly relates the model to the in vivo arrangement.

In vitro cell culture models of the blood-brain barrier (BBB) are important tools used to study cellular physiology and brain disease therapeutics. While many models exist, it is not clear whether any of these have been effectively optimized. The current work presents a sequential-screening study to optimize the culture conditions of an in vitro model of the blood-brain barrier.

The practice of medicine stands at the threshold of a transformation from its current focus on the treatment of disease events to an emphasis on enhancing health, preventing disease and personalizing care to meet each individual's specific health needs. Personalized health care is a new and strategic approach that is driven by personalized health planning empowered by personalized medicine tools, which are facilitated by advances in science and technology. These tools improve the capability to predict health risks, to determine and quantify the dynamics of disease development, and to target therapeutic approaches to the needs of the individual. Personalized health care can be implemented today using currently available technologies and know-how and thereby provide a market for the rational introduction of new personalized medicine tools. The need for early adoption of personalized health care stems from the necessity to reduce the egregious and wasteful burden of preventable chronic diseases, which is not effectively addressed by our current approach to care.

“Personalized Health Care” utilizes personalized health planning in conjunction with coordinated delivery systems to deliver personalized, predictive, preventive, and participatory care. This strategic approach to care is built on the concepts of systems biology and enables personalized medicine to be applied broadly to heath care delivery.

Cell-penetrating peptides (CPPs) are attractive vectors for in vivo and in vitro cellular uptake. Their use is, however, limited by insufficient understanding of their preference for a target cell. Here, a new CPP screening method is presented that uses mRNA display. After incubating the target cell lines, such as human embryonic kidney 293 (HEK 293) and HeLa cells, with an mRNA display library for 3 h at 37°C, the CPP-mRNA nucleotide conjugates were harvested. These were amplified with PCR and subsequently sequenced. The screened CPPs for each cell line were identified after four rounds of selection. Among them, two peptides, MAMPGEPRRANVMAHKLEPASLQLR NSCA (CPPK) and MAPQRDTVGGRTTPPSWGPAKAQLRNSCA (CPPL) were selected, and the FITC-labeled peptides were evaluated for their ability to penetrate cells. The screened CPPs were superior to polyarginine (R₁₁), which is widely used as a standard peptide and shows good cell penetration efficiency. Our method can be applied to other target cells for which CPPs have not yet been elucidated.

Drug delivery into the cell has been a major challenge for researchers in both academia and the pharmaceutical industry. Cell-penetrating peptides (CPPs) offer an attractive solution to this problem. In this article, the authors demonstrate that CPPs can be screened by mRNA display using a model cell line and also how this process can be applied to other target cell lines.

Biochemical and mechanical cues of the extracellular matrix have been shown to play important roles in cell-matrix and cell-cell interactions. We have experimentally tested the combined influence of these cues to better understand cell motility, force generation, cell-cell interaction, and assembly in an in vitro breast cancer model. MCF-10A non-tumorigenic mammary epithelial cells were observed on surfaces with varying fibronectin ligand concentration and polyacrylamide gel rigidity. Our data show that cell velocity is biphasic in both matrix rigidity and adhesiveness. The maximum cell migration velocity occurs only at specific combination of substrate stiffness and ligand density. We found cell-cell interactions reduce migration velocity. However, the traction forces cells exert onto the substrate increase linearly with both cues, with cells in pairs exerting higher maximum tractions observed over single cells. A relationship between force and motility shows a maximum in single cell velocity not observed in cell pairs. Cell-cell adhesion becomes strongly favored on softer gels with elasticity ≤1250 Pascals (Pa), implying the existence of a compliance threshold that promotes cell-cell over cell-matrix adhesion. Finally on gels with stiffness similar to pre-malignant breast tissue, 400 Pa, cells undergo multicellular assembly and division into 3D spherical aggregates on a 2D surface.

Understanding cell migration and cell-cell interactions are key to understanding cell invasion, a critical step in the progression of breast cancer. In this study, the authors show that the migration velocity and the assembly of breast cancer epithelial cells is optimal at intermediate concentrations of fibronectin concentration and substrate compliance. On low compliance polyacrylamide gels (400 Pa), cells assemble into clusters, whereas on stiffer gels, cells remain dispersed.

Systems biology has greatly contributed toward the analysis and understanding of biological systems under various genotypic and environmental conditions on a much larger scale than ever before. One of the applications of systems biology can be seen in unraveling and understanding complicated human diseases where the primary causes for a disease are often not clear. The in silico genome-scale metabolic network models can be employed for the analysis of diseases and for the discovery of novel drug targets suitable for treating the disease. Also, new antimicrobial targets can be discovered by analyzing, at the systems level, the genome-scale metabolic network of pathogenic microorganisms. Such applications are possible as these genome-scale metabolic network models contain extensive stoichiometric relationships among the metabolites constituting the organism's metabolism and information on the associated biophysical constraints. In this review, we highlight applications of genome-scale metabolic network modeling and simulations in predicting drug targets and designing potential strategies in combating pathogenic infection. Also, the use of metabolic network models in the systematic analysis of several human diseases is examined. Other computational and experimental approaches are discussed to complement the use of metabolic network models in the analysis of biological systems and to facilitate the drug discovery pipeline.

A nano-brous core-sheath structured scaffold incorporated with bioactive agents is supposed to promote cell migration, proliferation, and gene expressions through the controllable and sustainable release of bioactive agents from the fibers and the preservation of bioactivity. Here we present a novel and effective emulsion electrospinning method for obtaining fluorescein isothiocyanate-dextran (FITC-dextran)/poly(lactic-co-glycolic acid) (PLGA) and type I collagen/PLGA fibrous composite scaffolds. Core-sheath structured fibers with average diameters of 665 nm for FITC-dextran/PLGA and 567 nm for collagen/PLGA were successfully fabricated. In vitro-release profile shows sustained release of encapsulated FITC-dextran from FITC-dextran/PLGA fibers for as long as 7 weeks. The osteoblastic activity of the collagen/PLGA nanofibrous scaffold was investigated employing the osteoblastic-like MC3T3-E1 cell line. The results of the lactate dehydrogenase assay suggested excellent cytocompatibility. Cell proliferation and alkaline phosphatase activity were also ameliorated on this emulsion-electrospun collagen/PLGA fibrous scaffold. All the results indicated that this composite scaffold could support the early stages of osteoblast behavior as well as the immediate/late stages. The emulsion electrospinning process has potential for application in drug-release devices and as a 3-D scaffold in bone regeneration.

Microbiological production of glutathione using genetically engineered yeast strains has a potential to satisfy the increasing industrial demand of this tripeptide. In the present work accumulation of glutathione in response to YAP1 over-expression in Saccharomyces cerevisiae was studied. The over-expression resulted in intracellular glutathione level over two times higher than in the parent strain. Transcript analyses revealed that, in addition to the genes encoding enzymes in the glutathione biosynthesis pathway (GSH1 and GSH2), the expression levels of the genes in the cysteine biosynthesis pathway (CYS3 and CYS4) were also significantly higher in the YAP1 over-expressed strain. This suggests that YAP1 over-expression affects glutathione accumulation at both its biosynthesis and substrate availability levels.

Urea has been considered as a promising alternative nitrogen source for the cultivation of Arthrospira platensis if it is possible to avoid ammonia toxicity; however, this procedure can lead to periods of nitrogen shortage. This study shows that the addition of potassium nitrate, which acts as a nitrogen reservoir, to cultivations carried out with urea in a fed-batch process can increase the maximum cell concentration (X_m) and also cell productivity (P_X). Using response surface methodology, the model indicates that the estimated optimum X_m can be achieved with 17.3 mM potassium nitrate and 8.9 mM urea. Under this condition an X_m of 6077 ± 199 mg/L and a P_X of 341.5 ± 19.1 mg L^–1day^–1 were obtained.

Modification of proteins with small molecules is a widely used and powerful tool in biological research. Enzymatic approaches are particularly promising because substrate specificity allows for site-specific modification. Sortase A, a transpeptidase from Staphylococcus aureus, cleaves between the T and G residues in the sequence LPXTG, and subsequently links the carboxyl group of the T residue to an amino group of N-terminal glycine oligomers by a native peptide bond. Although Gram-positive bacteria have several kinds of sortases, there are few reports concerning their expression and substrate specificity. Here, we demonstrate site-specific protein modification with primary amine-containing molecules catalyzed by Lactobacillus plantarum sortase. Enhanced green fluorescent protein (EGFP) was employed as a model protein, and an amine-containing biotin molecule was site-specifically conjugated with LPQTSEQ-tagged EGFP. We developed a novel Lactobacillus plantarum sortase that has different substrate specificity compared to Staphylococcus aureus sortase. Amine-directed protein modification was achieved using the Lactobacillus plantarum sortase ''LPQTSEQ'' sequence original recognition tag. Our results demonstrate a promising method for expanding the capabilities of site-specific protein-small molecule modification.

Nature takes advantage of the malleability of protein and RNA sequence and structure to employ these macromolecules as molecular reporters whose conformation and functional roles depend on the presence of a specific ligand (an “effector” molecule). By following nature's example, ligand-responsive proteins and RNA molecules are now routinely engineered and incorporated into customized molecular reporting systems (biosensors). Microbial small-molecule biosensors and endogenous molecular reporters based on these sensing components find a variety of applications that include high-throughput screening of biosynthesis libraries, environmental monitoring, and novel gene regulation in synthetic biology. Here, we review recent advances in engineering small-molecule recognition by proteins and RNA and in coupling in vivo ligand binding to reporter-gene expression or to allosteric activation of a protein conferring a detectable phenotype. Emphasis is placed on microbial screening systems that serve as molecular reporters and facilitate engineering the ligand-binding component to recognize new molecules.

Fetal liver epithelial cells (FLEC) are valuable for liver cell therapy and tissue engineering, but methods for culture and characterization of these cells are not well developed. This work explores the influence of multiple soluble factors on FLEC, with the long-term goal of developing an optimal culture system to generate functional liver tissue. Our comparative analysis suggests hepatocyte growth factor (HGF) is required throughout the culture period. In the presence of HGF, addition of oncostatin M (OSM) at culture initiation results in concurrent growth and maturation, while constant presence of protective agents like ascorbic acid enhances cell survival. Study observations led to the development of a culture medium that provided optimal growth and hepatic differentiation conditions. FLEC expansion was observed to be approximately twofold of that under standard conditions, albumin secretion rate was 2–3 times greater than maximal values obtained with other media, and the highest level of glycogen accumulation among all conditions was observed with the developed medium. Our findings serve to advance culture methods for liver progenitors in cell therapy and tissue engineering applications.

Precise histopathological localization of E-cadherin and p63 is of immense importance in understanding the integrity of oral mucosal stratified epithelium in normal and diseased conditions. Necessarily immunohistochemical imaging should have minimum bleaching impact on the dyes and ability to produce clear and crisp images. Here ApoTome provides an alternative with metal halide light source and structured illumination under the assistance of grids, along with integrated image processing modality to generate crisp images with digital interface. The current study demonstrates the applicability of such microscopic system in capturing fluorescence images of immunohistochemical sections of normal and precancerous biopsies in respect to the expression of p63 and E-cadherin in the epithelial cells. The ApoTome images localize the nuclear and membranous expressions of p63 and E-cadherin, respectively, with remarkable specificity. The findings on E-cadherin expression have enormous diagnostic significance as these images clearly differentiate the early and advanced stages of oral submucous fibrosis based on their cytoplasmic and membranous location. Thus, this study clearly depicts a remarkable performance of ApoTome with diagnostic significance.

Previous mathematical modeling efforts have made significant contributions to the development of systems biology for predicting biological behavior quantitatively. However, dynamic metabolic model construction remains challenging due to uncertainties in mechanistic structures and parameters. In addition, parameter estimation and model validation often require designated experiments conducted only for purpose of modeling. Such difficulties have hampered the progress of modeling in biology and biotechnology. To circumvent these problems, ensemble approaches have been used to account for uncertainties in model structure and parameters. Specifically, this review focuses on approaches that utilize readily available fermentation data for parameter screening and model validation. Time course data for metabolite measurements, if available, can further calibrate the model. The basis for this approach is explained in non-mathematical terms accessible to experimentalists. Information gained from such an approach has been shown to be useful in designing Escherichia coli strains for metabolic engineering and synthetic biology.

Type I and II secretory pathways are used for the translocation of recombinant proteins from the cytoplasm of Escherichia coli. The purpose of this study was to evaluate four signal peptides (HlyA, TorA, GeneIII, and PelB), representing the most common secretion pathways in E. coli, for their ability to target green fluorescent protein (GFP) for membrane translocation. Signal peptide-GFP genetic fusions were designed in accordance with BioFusion standards (BBF RFC 10, BBF RFC 23). The HlyA signal peptide targeted GFP for secretion to the extracellular media via the type I secretory pathway, whereas TAT-dependent signal peptide TorA and Sec-dependent signal peptide GeneIII exported GFP to the periplasm. The PelB signal peptide was inefficient in translocating GFP. The use of biological technical standards simplified the design and construction of functional signal peptide-recombinant protein genetic devices for type I and II secretion in E. coli. The utility of the standardized parts model is further illustrated as constructed biological parts are available for direct application to other studies on recombinant protein translocation.

There is increasing interest in bioengineering of lipids for use in functional foods, pharmaceuticals, and biofuels. Saccharomyces cerevisiae is a widely utilized cell factory for biotechnological production, thus a tempting alternative. Herein, we show how its neutral lipid accumulation varies throughout metabolic phases under nutritional conditions relevant for large-scale fermentation. Population-averaged metabolic data were correlated with lipid storage at the single-cell level monitored at submicron resolution by label-free coherent anti-Stokes Raman scattering (CARS) microscopy. While lipid droplet sizes are fairly constant, the number of droplets is a dynamic parameter determined by glucose and ethanol levels. The lowest number of lipid droplets is observed in the transition phase between glucose and ethanol fermentation. It is followed by a buildup during the ethanol phase. The surplus of accumulated lipids is then mobilized at concurrent glucose and ethanol starvation in the subsequent stationary phase. Thus, the highest amount of lipids is found in the ethanol phase, which is about 0.3 fL/cell. Our results indicate that the budding yeast, S. cerevisiae, can be used for the biosynthesis of lipids and demonstrate the strength of CARS microscopy for monitoring the dynamics of lipid metabolism at the single-cell level of importance for optimized lipid production.

Assuring the microbiological safety of biological therapeutics remains an important concern. Our group has recently reported small trimeric peptides that have the ability to bind and remove a model nonenveloped virus, porcine parvovirus (PPV), from complex solutions containing human blood plasma. In an effort to improve the removal efficiency of these small peptides, we created a biased library of hexamer peptides that contains two previously reported trimeric peptides designated WRW and KYY. This library was screened and several hexamer peptides were discovered that also removed PPV from solution, but there was no marked improvement in removal efficiency when compared to the trimeric peptides. Based on simulated docking experiments, it appeared that hexamer peptide binding is dictated more by secondary structure, whereas the binding of trimeric peptides is dominated by charge and hydrophobicity. This study demonstrates that trimeric and hexameric peptides may have different, matrix-specific roles to play in virus removal applications. In general, the hexamer ligand may perform better for binding of specific viruses, whereas the trimer ligand may have more broadly reactive virus-binding properties.

Microalgae can be used to produce versatile high-value fuels, such as methane, biodiesel, ethanol, or hydrogen gas. One of the most important factors that influence the economics of microalgae cultivation is the primary production of biomass per unit area. This is determined by productivity rates during cultivation, which are influenced by the local climate conditions (solar irradiation, temperature). To compare locations in different climate regions for microalgae cultivation, a mathematical model for an idealized closed photobioreactor was developed. The applied growth kinetics were based on theoretical maximum photon-conversion efficiencies (for the conversion of solar energy to chemical energy in the form of biomass). Known or estimated temperature effects for different algal strains were incorporated. The model was used to calculate hourly average areal productivity rates as well as annual primary production values under local conditions at seven example locations. Here, hourly weather data (solar irradiance and air temperature) were taken into account. According to these model calculations, maximum annual yields were achieved in regions with high irradiation and temperature patterns in or near the optimum range of the specific algal strain (here, desert and equatorial humid climates). The developed model can be used as a tool to assess and compare individual locations for microalgae cultivation.

In spite of the importance of Chinese hamster ovary (CHO) cells for recombinant protein production, very little is known about the molecular and gene regulatory mechanisms that control cellular phenotypes such as enhanced growth under serum-free conditions or high productivity. Most microarray analyses to this purpose are performed with samples taken during the exponential growth phase. However, the cellular transcriptome is dynamic, changing in response to external and internal stimuli and thus reflecting the current functional capacity of cells as well as their ability to adapt to a changing environment. Therefore, during batch or fed-batch cultivations it can be expected that the transcription pattern of genes will change and that such changes may give indications on the cellular state in terms of viability, growth, and productivity. In the current study we monitored the change in expression patterns of mRNAs and microRNAs (miRNA) during lag, exponential, and stationary phases in CHO-K1 suspension cell cultures. In total, over 1400 mRNAs and more than 100 miRNAs were differentially regulated (p<0.05) relative to the batch culture at the starting point. Functional clustering revealed groups of genes with similar expression patterns, which were subjected to functional pathway analysis. In addition, as miRNAs generally act as negative post-transcriptional regulators of mRNAs, we looked for changes in their expression that were inverse to those of their predicted target mRNAs.

In the current work we demonstrate the relevance of monochromatic light conditions in moss plant cell culture. Light intensity and illumination wavelength are important cultivation parameters due to their impact on growth and chlorophyll formation kinetics of the moss Physcomitrella patens. This moss was chosen as a model organism due to its capability to produce complex recombinant pharmaceutical proteins. Filamentous moss cells were cultivated in mineral medium in shaking flasks. The flasks were illuminated by light emitting diodes (LED) providing nearly monochromatic red and blue light as well as white light as a reference. A maximum growth rate of 0.78 day⁽¹ was achieved under additional CO₂ aeration and no growth inhibition was observed under high light illumination. The application of dual red and blue light is the most effective way to reach high growth and chlorophyll formation rates while minimizing energy consumption of the LEDs. These observations are discussed as effects of photo sensory pigments in the moss. The combination of monochromatic red and blue light should be considered when a large scale process is set up.

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Industrial biotechnology. Renewable biomass can be used as a raw material for the production of various chemicals, fuels and materials in a cost-effective manner by employing metabolically engineered high performance microorganisms. This cover art shows production of biobutanol from renewable biomass by employing engineered clostridia (see the article by Jang et al. http://dx.doi.org/10.1002/biot.201100059). The cover picture was created by Prof. Sang Yup Lee and Dr. Yu Sin Jang at KAIST, Korea.

Biofuel from renewable biomass is one of the answers to help solve the problems associated with limited fossil resources and climate change. Butanol has superior liquid-fuel characteristics, with similar properties to gasoline, and thus, has the potential to be used as a substitute for gasoline. Clostridia are recognized as a good butanol producers and are employed in the industrial-scale production of solvents. Due to the difficulty of performing genetic manipulations on clostridia, however, strain improvement has been rather slow. Furthermore, complex metabolic characteristics of acidogenesis followed by solventogenesis in this strain have hampered the development of engineered clostridia strains with highly efficient and selective butanol-production capabilities. In recent years, the butanol-producing characteristics in clostridia have been further characterized and alternative pathways discovered. More recently, systems-level metabolic engineering approaches were taken to develop superior strains. Herein, we review recent discoveries of metabolic pathways for butanol production and the metabolic engineering strategies being developed.

Biofuel from renewable biomass is one of the solutions to the limited supply of fossil resources and climate change. Butanol has superior liquid-fuel characteristics, with similar properties to gasoline, and thus, has the potential to be used as a substitute for gasoline. Herein, we review recent discoveries of metabolic pathways for butanol production from biomass and the metabolic engineering strategies being developed.

Due to increasing concerns about environmental problems, climate change and limited fossil resources, bio-based production of chemicals and polymers is gaining attention as one of the solutions to these problems. Polyhydroxyalkanoates (PHAs) are polyesters that can be produced by microbial fermentation. PHAs are synthesized using monomer precursors provided from diverse metabolic pathways and are accumulated as distinct granules inside the cells. On the other hand, most so-called bio-based polymers including polybutylene succinate, polytrimethylene terephthalate, and polylactic acid (PLA) are synthesized by a chemical process using monomers produced by fermentation. PLA, an attractive biomass-derived plastic, is currently synthesized by heavy metal-catalyzed ring opening polymerization of L-lactide that is made from fermentation-derived L-lactic acid. Recently, a complete biological process for the production of PLA and PLA copolymers from renewable resources has been developed by direct fermentation of recombinant bacteria employing PHA biosynthetic pathways coupled with a novel metabolic pathway. This could be accomplished by establishing a pathway for generating lactyl-CoA and engineering PHA synthase to accept lactyl-CoA as a substrate combined with systems metabolic engineering. In this article, we review recent advances in the production of lactate-containing homo- and co-polyesters. Challenges remaining to efficiently produce PLA and its copolymers and strategies to overcome these challenges through metabolic engineering combined with enzyme engineering are discussed.

This review article discusses recent advances in the production of lactate-containing homo- and co-polyesters by metabolically engineered bacteria. The manuscript highlights challenges remaining to efficiently produce PLA and its copolymers and strategies to overcome these challenges through metabolic engineering combined with enzyme engineering.

Succinate has been recognized as an important platform chemical that can be produced from biomass. While a number of organisms are capable of succinate production naturally, this review focuses on the engineering of Escherichia coli for the production of four-carbon dicarboxylic acid. Important features of a succinate production system are to achieve an optimal balance of reducing equivalents generated by consumption of the feedstock, while maximizing the amount of carbon channeled into the product. Aerobic and anaerobic production strains have been developed and applied to production from glucose and other abundant carbon sources. Metabolic engineering methods and strain evolution have been used and supplemented by the recent application of systems biology and in silico modeling tools to construct optimal production strains. The metabolic capacity of the production strain, the requirement for efficient recovery of succinate, and the reliability of the performance under scaleup are important in the overall process. The costs of the overall biorefinery-compatible process will determine the economic commercialization of succinate and its impact in larger chemical markets.

Bio-based succinate production from sugar feedstocks using engineered E. coli: The metabolism of the bacterium, Escherichia coli has been engineered to produce succinic acid from a variety of commonly available feedstocks. This molecule is valuable in specialized applications and can be converted to a variety of other industrial chemicals and polymers by known processes. This review focuses on the engineering of metabolic pathways within E. coli for improved succinic acid production.

Industrial production of β-lactam antibiotics by the filamentous fungus Penicillium chrysogenum is based on successive classical strain improvement cycles. This review summarizes our current knowledge on the results of this classical strain improvement process, and discusses avenues to improve β-lactam biosynthesis and to exploit P. chrysogenum as an industrial host for the production of other antibiotics and peptide products. Genomic and transcriptional analysis of strain lineages has led to the identification of several important alterations in high-yielding strains, including the amplification of the penicillin biosynthetic gene cluster, elevated transcription of genes involved in biosynthesis of penicillin and amino acid precursors, and genes encoding microbody proliferation factors. In recent years, successful metabolic engineering and synthetic biology approaches have resulted in the redirection of the penicillin pathway towards the production of cephalosporins. This sets a new direction in industrial antibiotics productions towards more sustainable methods for the fermentative production of unnatural antibiotics and related compounds.

The filamentous fungus Penicillium chrysogenum is used for the industrial production of β-lactam antibiotics such as penicillin G. Through advanced metabolic engineering and synthetic biology approaches, the penicillin biosynthesis pathway can be redirected towards the production of cephalosporins and penicillins that are normally produced through semi-synthetic means. This technology now enables more sustainable methods for the fermentative production of unnatural antibiotics and related compounds.

This review provides an overview of the properties, different biosynthetic machineries, and biotechnological production processes of four microbially derived glucuronic acid-based polysaccharides that are of interest for diverse biomedical purposes. In particular, the utilization of hyaluronic acid and heparin sulfate in high-value medical applications is already well established, whereas chondroitin sulfate and alginate show high potential within this ever-growing field. Furthermore, new strategies exploiting genetically engineered microorganisms generated through improving naturally existing pathways or de novo designed ones are described. These new developments result in increased fermentation titers, and thereby, pave the way towards feasible, or at least improved, process economy. Moreover, these strategies also allow for the future possibility of producing tailor-made biopolymers with specified characteristics, even novel molecules.

A safer alternative to animal tissue extraction or chemical synthesis of biomedically relevant polymers is the development of biotech processes that exploit capsular/extracellular polysaccharide-producing microorganisms. This review provides an overview on recent advances in the development of molecular tools and fermentation technologies that lead to a better understanding of the metabolic mechanisms and required process conditions for microbial production of four glucuronic acid-based polymers (HA, CS, heparin, alginate).

Plasmid DNA (pDNA) has become very attractive as a biopharmaceutical, especially for gene therapy and DNA vaccination. Currently, there are a few products licensed for veterinary applications and numerous plasmids in clinical trials for use in humans. Recent work in both academia and industry demonstrates a need for technological and economical improvement in pDNA manufacturing. Significant progress has been achieved in plasmid design and downstream processing, but there is still a demand for improved production strains. This review focuses on engineering of Escherichia coli strains for plasmid DNA production, understanding the differences between the traditional use of pDNA for recombinant protein production and its role as a biopharmaceutical. We will present recent developments in engineering of E. coli strains, highlight essential genes for improvement of pDNA yield and quality, and analyze the impact of various process strategies on gene expression in pDNA production strains.

Plasmid DNA (pDNA) has the potential to be an effective vector for gene therapy and DNA vaccination. Remarkable progress has been made in plasmid design and downstream processing, but there is still a demand for improved production strains. This review focuses on recent work related to engineering of Escherichia coli host strains for plasmid DNA production. As shown in the graphical abstract, the review emphasizes the interplay between the many factors that influence strain design.

Downstream processing of chitosan requires several technological steps that contribute to the total production costs. Precipitation and especially evaporation are energy-consuming processes, resulting in higher costs and limiting industrial scale production. This study investigated the filtration kinetics of chitosan derived from cell walls of fungi and from exoskeletons of arthropods by electrofiltration, an alternative method, thus reducing the downstream processing steps and costs. Experiments with different voltages and pressures were conducted in order to demonstrate the effect of both parameters on filtration kinetics. The concentration of the biopolymer was obtained by the average factor of 40 by applying an electric field of 4 V/mm and pressure of 4 bars. A series of analytical experiments demonstrated the lack of structural and functional changes in chitosan molecules after electrofiltration. These results, combined with the reduction of energy and processing time, define the investigated method as a promising downstream step in the chitosan production technology.

Downstream processing of chitosan requires several technological steps that contribute to the total production costs. Precipitation and especially evaporation are energy-consuming processes, resulting in higher costs and limiting industrial scale production. In this study, the authors investigated the filtration kinetics of chitosan derived from cell walls of fungi and from exoskeletons of arthropods by electrofiltration, thus reducing the downstream processing steps and costs.

We analysed the influence of several enzymatic treatment processes using an alkaline cellulase enzyme from Bacillus spp. on the sorption properties of cotton fabrics. Although cellulases are commonly applied in detergent formulations due to their anti-redeposition and depilling benefits, determining the mechanism of action of alkaline cellulases on cotton fibres requires a deeper understanding of the morphology and structure of cotton fibres in terms of fibre cleaning. The accessibility of cellulose fibres was studied by evaluating the iodine sorption value and by fluorescent-labelled enzyme microscopy; the surface morphology of fabrics was analysed by scanning microscopy. The action of enzyme hydrolysis over short time periods can produce fibrillation on cotton fibre surface without any release of cellulosic material. The results indicate that several short consecutive treatments were more effective in increasing the fibre accessibility than one long treatment. In addition, no detectable hydrolytic activity, in terms of reducing sugar production, was found.

Cellulases have been applied in detergent formulations for the last 30 years. To fully understand the mechanism of how alkaline cellulases act on cotton fibers a deeper understanding of the structure of cotton fibers during cleaning is required. In this study, the authors have analyzed the influence of several enzymatic treatment processes using an alkaline cellulase enzyme from Bacillus spp. on the sorption properties of cotton fabrics. Several short consecutive treatments were more effective in accessing the fibers while no hydrolytic activity, i.e sugar production was detected.

This study combines the properties of siloxanes and lignin polymers to produce hybrid functional polymers that can be used as adhesives, coating materials, and/or multifunctionalized thin-coating films. Lignin-silica hybrid copolymers were synthesized by using a sol-gel process. Laccases from Trametes hirsuta were used to oxidize lignosulphonates to enhance their reactivity towards siloxanes and then were incorporated into siloxane precursors undergoing a sol-gel process. In vitro copolymerization studies using pure lignin monomers with aminosilanes or ethoxytrimethylsilane and analysis by ²⁹Si NMR spectroscopy revealed hybrid products. Except for kraft lignin, an increase in lignin concentration positively affected the tensile strength in all samples. Similarly, the viscosity generally increased in all samples with increasing lignin concentration and also affected the curing time.

Lignin is currently extensively investigated as a raw material for existing and novel hybrid polymers. The multicopper-containing enzymes, Laccases (benzenediol: oxygen oxidoreductases, EC.1.10.3.2), play a major role in activating lignin by oxidation, thereby increasing the interaction between lignin and siloxane precursors, resulting in interpenetrating polymers. In this article, the authors synthesize lignin-siloxane hybrid coating films to produce hybrid functional polymers that can be used as adhesives, coating materials, and/or multifunctionalized thin-coating films.

The main hindrance in the use of polyhydroxyalkanoates (PHAs) as a replacement for existing petroleum-based plastics is their high production cost. The carbon source accounts for 50% of the cost for PHA production. Thus, increasing the yield and productivity of PHAs on cheap substrates is an important challenge for biotechnologists to support the commercialization and further applications of these polymers. In this study, we have investigated the use of an agricultural raw material, sugarcane molasses, as the main carbon source for poly(3-hydroxybutyrate) (P(3HB)) production by Bacillus cereus SPV. These studies were carried out in both shaken flasks and 2 L bioreactors. Various conditions were evaluated for their effects on biomass and P(3HB) accumulation. A high polymer yield was obtained, 61.07% dry cell weight (DCW) in a 1 L shaken flask study and 51.37% DCW in a 2 L fermenter study. These yields are 50% higher than previously observed with Bacillus cereus SPV. Hence, the results are encouraging and show that sugarcane molasses are a promising carbon source for an economical and commercially viable production of P(3HB).

Sugarcane molasses are a promising carbon source for an economical and commercially viable production of P(3HB). The main hindrance in the use of polyhydroxyalkanoates (PHAs) as a replacement for existing petroleum-based plastics is their high production cost. In this study, the authors investigate the use of an agricultural raw material, sugarcane molasses, as the main carbon source for poly (3-hydroxybutyrate) (P(3HB)) production by Bacillus cereus SPV. The polymer yield was 50% higher than previously reported with the same organism.

In the current context of global warming, the substitution of conventional plastics with bioplastics is a challenge. To take up this challenge, we must meet different technical and economic constraints. In the case of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the technical properties can be modulated by varying the 3-hydroxyvalerate content. 3-Hydroxyvalerate (3-HV) enhancement is an issue; therefore, simultaneous evaluation of several 3-hydroxyvalerate-enhancing substrates through fractional factorial design of experiments is described. Eight substrates citric, valeric, propionic, and levulinic acids; propanol; pentanol; and sodium propionate were studied for 3-HV enhancement, and sodium glutamate was studied for biomass and polyhydroxyalkanoate (PHA) enhancement. The most efficient 3-hydroxyvalerate-enhancing factors were levulinic acid, sodium propionate, and pentanol; however, pentanol, at a concentration of 1 g/L, had an extremely negative influence on biomass production and the PHA content of cells. The effect of the inoculum nutrient composition on the final 3-HVcontent was also evaluated. These results showed that the most efficient combination for the production of high 3-HVcontent in PHBV was primary inoculum growth on mineral medium followed by fermentation for 48 h with levulinic acid and sodium propionate (at 1 g/L) as the only carbon sources. This allowed us to produce PHBV with a 3-HVcontent of 80 mol % and overall volumetric and specific productivities of 2 mg/L/h and 3.9 mg/g_CDW/h, respectively, with the addition of only 2 g/L of inducing substances.

Polyhydroxyalkanoates (PHAs) are a family of bioplastics with a very wide range of properties and applications. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is among the most popular PHAs, because the 3-hydroxyvalerate (3-HV) content improves the thermal and mechanical properties of PHBV. This article reports the enhancement of the 3-HV component in PHBV production by Cupriavidus necator.