Showing papers in "Biotechnology Journal in 2019"

Yeast Systems Biology: Model Organism and Cell Factory.

Abstract: For thousands of years, the yeast Saccharomyces cerevisiae (S. cerevisiae) has served as a cell factory for the production of bread, beer, and wine. In more recent years, this yeast has also served as a cell factory for producing many different fuels, chemicals, food ingredients, and pharmaceuticals. S. cerevisiae, however, has also served as a very important model organism for studying eukaryal biology, and even today many new discoveries, important for the treatment of human diseases, are made using this yeast as a model organism. Here a brief review of the use of S. cerevisiae as a model organism for studying eukaryal biology, its use as a cell factory, and how advances in systems biology underpin developments in both these areas, is provided.

Identification and Antibiotic‐Susceptibility Profiling of Infectious Bacterial Agents: A Review of Current and Future Trends

Abstract: Antimicrobial resistance is one of the most worrying threats to humankind with extremely high healthcare costs associated. The current technologies used in clinical microbiology to identify the bacterial agent and profile antimicrobial susceptibility are time-consuming and frequently expensive. As a result, physicians prescribe empirical antimicrobial therapies. This scenario is often the cause of therapeutic failures, causing higher mortality rates and healthcare costs, as well as the emergence and spread of antibiotic resistant bacteria. As such, new technologies for rapid identification of the pathogen and antimicrobial susceptibility testing are needed. This review summarizes the current technologies, and the promising emerging and future alternatives for the identification and profiling of antimicrobial resistance bacterial agents, which are expected to revolutionize the field of clinical diagnostics.

Latest Advances on Bacterial Cellulose-Based Materials for Wound Healing, Delivery Systems, and Tissue Engineering.

Abstract: Bacterial cellulose (BC) is a nanocellulose form produced by some nonpathogenic bacteria. BC presents unique physical, chemical, and biological properties that make it a very versatile material and has found application in several fields, namely in food industry, cosmetics, and biomedicine. This review overviews the latest state-of-the-art usage of BC on three important areas of the biomedical field, namely delivery systems, wound dressing and healing materials, and tissue engineering for regenerative medicine. BC will be reviewed as a promising biopolymer for the design and development of innovative materials for the mentioned applications. Overall, BC is shown to be an effective and versatile carrier for delivery systems, a safe and multicustomizable patch or graft for wound dressing and healing applications, and a material that can be further tuned to better adjust for each tissue engineering application, by using different methods.

Implementation of Fully Integrated Continuous Antibody Processing: Effects on Productivity and COGm

Abstract: The changing landscape of the biopharmaceutical market is driving a paradigm shift toward continuous manufacturing. To date, integrated continuous bioprocessing has not been realized as enabling technologies are nascent. In this work, a fully integrated continuous process is successfully demonstrated from pilot scale bioreactor to drug substance. Comparable product quality is observed between the continuous process and a 500 L fed-batch conventional process. The continuous process generated material at a rate of 1 kg of purified mAb every 4 days, achieving a 4.6-fold increase in productivity compared to the fed-batch process A plant throughput analysis using BioSolve software shows that a fed-batch facility with 4 × 12 500 L stainless steel bioreactors and purification train of the corresponding scale can be replaced by a continuous facility consisting of 5 × 2000 L single use bioreactors and smaller purification train, with a cost reduction of 15%.

Bioeconomy for Sustainable Development.

Abstract: Bioeconomy is an emerging paradigm under which the creation, development, and revitalization of economic systems based on a sustainable use of renewable biological resources in a balanced way is rapidly spreading globally. Bioeconomy is building bridges between biotechnology and economy as well as between science, industry, and society. Biotechnology, from its ancient origins up to the present is at the core of the scientific and innovative foundation of bioeconomy policies developed in numerous countries. The challenges and perspectives of bioeconomies are immense, from resource-efficient large-scale manufacturing of products such as chemicals, materials, food, pharmaceuticals, polymers, flavors, and fragrances to the production of new biomaterials and bioenergy in a sustainable and economic way for a growing world population. Key success factors for different countries working on the bioeconomy vary widely from high-tech bioeconomies, emerging diversified or diversified bioeconomies to advanced and basic primary sector bioeconomies. Despite the large variety of bioeconomies, several common elements are identified, which are simultaneously needed altogether.

Biotechnology, Big Data and Artificial Intelligence.

Abstract: Developments in biotechnology are increasingly dependent on the extensive use of big data, generated by modern high-throughput instrumentation technologies, and stored in thousands of databases, public and private. Future developments in this area depend, critically, on the ability of biotechnology researchers to master the skills required to effectively integrate their own contributions with the large amounts of information available in these databases. This article offers a perspective of the relations that exist between the fields of big data and biotechnology, including the related technologies of artificial intelligence and machine learning and describes how data integration, data exploitation, and process optimization correspond to three essential steps in any future biotechnology project. The article also lists a number of application areas where the ability to use big data will become a key factor, including drug discovery, drug recycling, drug safety, functional and structural genomics, proteomics, pharmacogenetics, and pharmacogenomics, among others.

3D Primary Hepatocyte Culture Systems for Analyses of Liver Diseases, Drug Metabolism, and Toxicity : Emerging Culture Paradigms and Applications

Abstract: Recent research has shown that the maintenance of relevant liver functions ex vivo requires models in which the cells exhibit an in vivo-like phenotype, often achieved by reconstitution of appropriate cellular interactions. Multiple different models have been presented that differ in the cells utilized, media, and culture conditions. Furthermore, several technologically different approaches have been presented including bioreactors, chips, and plate-based systems in fluidic or static media constituting of chemically diverse materials. Using such models, the ability to predict drug metabolism, drug toxicity, and liver functionality have increased tremendously as compared to conventional in vitro models in which cells are cultured as 2D monolayers. Here, the authors highlight important considerations for microphysiological systems for primary hepatocyte culture, review current culture paradigms, and discuss their opportunities for studies of drug metabolism, hepatotoxicity, liver biology, and disease.

Scalable Production and Isolation of Extracellular Vesicles: Available Sources and Lessons from Current Industrial Bioprocesses.

Abstract: Potential applications of extracellular vesicles (EVs) are attracting increasing interest in the fields of medicine, cosmetics, and nutrition. However, the manufacturing of EVs is currently characterized by low yields. This limitation severely hampers progress in research at the laboratory and clinical scales, as well as the realization of successful and cost-effective EV-based products. Moreover, the high level of heterogeneity of EVs further complicates reproducible manufacturing on a large scale. In this review, possible directions toward the scalable production of EVs are discussed. In particular, two strategies are considered: i) the optimization of upstream unit operations and ii) the exploitation of well-established and mature technologies already in use in other industrial bioprocesses.

Genome Editing with CRISPR-Cas9 in Lactobacillus plantarum Revealed That Editing Outcomes Can Vary Across Strains and Between Methods.

Abstract: Lactic-acid bacteria such as Lactobacillus plantarum are commonly used for fermenting foods and as probiotics, where increasingly sophisticated genome-editing tools are employed to elucidate and enhance these microbes' beneficial properties The most advanced tools to date utilize an oligonucleotide or double-stranded DNA donor for recombineering and Cas9 for targeted DNA cleavage As the associated methods are often developed in isolation for one strain, it remains unclear how different Cas9-based editing methods compare across strains Here, this work directly compares two methods in different strains of L plantarum: one utilizing a plasmid-encoded recombineering template and another utilizing an oligonucleotide donor and an inducible DNA recombinase This comparison reveals one instance in which only the recombineering-template method generates desired edits and another instance in which only the oligo method generates desired edits It is further found that both methods exhibit highly variable success editing the same site across multiple L plantarum strains Finally, failure modes are identified for the recombineering-template method, including a consistent genomic deletion and reversion of a point mutation in the recombineering template This study therefore highlights surprising differences for Cas9-mediated genome editing between methods and related strains, arguing for the need for multiple, distinct methods when performing CRISPR-based editing in bacteria

Modeling the Downstream Processing of Monoclonal Antibodies Reveals Cost Advantages for Continuous Methods for a Broad Range of Manufacturing Scales.

Abstract: The biopharmaceutical industry is evolving in response to changing market conditions, including increasing competition and growing pressures to reduce costs. Single-use (SU) technologies and continuous bioprocessing have attracted attention as potential facilitators of cost-optimized manufacturing for monoclonal antibodies. While disposable bioprocessing has been adopted at many scales of manufacturing, continuous bioprocessing has yet to reach the same level of implementation. In this study, the cost of goods of Pall Life Science's integrated, continuous bioprocessing (ICB) platform is modeled, along with that of purification processes in stainless-steel and SU batch formats. All three models include costs associated with downstream processing only. Evaluation of the models across a broad range of clinical and commercial scenarios reveal that the cost savings gained by switching from stainless-steel to SU batch processing are often amplified by continuous operation. The continuous platform exhibits the lowest cost of goods across 78% of all scenarios modeled here, with the SU batch process having the lowest costs in the rest of the cases. The relative savings demonstrated by the continuous process are greatest at the highest feed titers and volumes. These findings indicate that existing and imminent continuous technologies and equipment can become key enablers for more cost effective manufacturing of biopharmaceuticals.

Extracellular Vesicles in Oncology: Progress and Pitfalls in the Methods of Isolation and Analysis.

Abstract: The possibility to study solid tumors through the analysis of extracellular vesicles in biological fluids is one of the most exciting and rapidly advancing field in cancer research. The extracellular vesicles are tiny sacs released in both physiological and pathological conditions and can be used to monitor the evolution of several pathological states, including neoplastic diseases. Indeed, these vesicles carry biological informations and can affect the behavior of recipient cells by transferring proteins, DNA, RNA, and microRNA. In this review the authors analyze the methods to collect biological fluid samples (urine, plasma/serum, and cell supernatant), and to isolate and quantify extracellular vesicles highlighting advantages and drawbacks. Moreover, the authors provide an overview on the adoption and the advantages of the methods (such as digital PCR, next generation sequencing, reverse-phase protein microarrays, flow-cytometry, etc.) most frequently used to analyze the molecular content of extracellular vesicles. Despite the great scientific interest on this topic, there is still a great uncertainty about which is the best method for the collection, isolation, quantification, and molecular evaluation of these vesicles and a standardization is needed. The features of EVs make them ideal candidates for liquid biopsy-based biomarkers. However, the small size of EVs makes their analysis very difficult and requires multiple advanced technologies, being therefore a limitation.

Enhanced Saccharification and Fermentation of Rice Straw by Reducing the Concentration of Phenolic Compounds Using an Immobilized Enzyme Cocktail

Abstract: The production of bioethanol from rice straw can contribute to the rural economy and provide clean fuel in a sustainable manner. However, phenolic compounds, which are mostly produced during acid pretreatment of biomass, act as inhibitors of fermenting microorganisms. Laccase is well known for its ability to oxidize lignin and phenolic compounds derived from lignocellulosic biomass. In the present study, an immobilized enzyme cocktail containing laccase was isevaluated in regard to its ability to enhance the saccharification and fermentation processes by reducing the amount of phenolic compounds produced. Saccharification of rice straw with the laccase-supplemented immobilized enzyme cocktail reduced phenolic compounds by 73.8%, resulting in a saccharification yield of 84.6%. In addition, improved yeast performance was is noted during the fermentation process, resulting in a 78.3% conversion of sugar into ethanol with an ethanol productivity of 0.478 g/L/h. To the best of our knowledge, this is the first description of the use of an immobilized enzyme cocktail comprised of Celluclast 1.5L, β-glucosidase, and laccase for the production of bioethanol from rice straw. This study details a potential approach to producing biofuels from agricultural biomass, the applicability of which can be improved through up-scaling.

Development of a RecE/T-Assisted CRISPR-Cas9 Toolbox for Lactobacillus

Abstract: Lactobacilli are members of a large family involved in industrial food fermentation, therapeutics, and health promotion. However, the development of genetic manipulation tools for this genus lags behind its relative industrial and medical significance. The development of clustered regularly interspaced short palindromic repeat (CRISPR)-based genome engineering for Lactobacillus is now underway. However, some Lactobacillus species are sensitive to CRISPR-Cas9 induced double strand breaks (DSBs) due to a deficiency in homology-directed repair (HDR), which allows chromosomal genetic editing. Here, phage-derived RecE/T is coupled with CRISPR-Cas9 and the transcriptional activity of broad-spectrum host promoters is assessed to set up a versatile toolbox containing a recombination helper plasmid and a broad host CRISPR-Cas9 editing plasmid, which enables efficient genome editing in Lactobacillus plantarum (L. plantarum) WCFS1 and Lactobacillus brevis (L. brevis) ATCC367. The RecE/T-assisted CRISPR-Cas9 toolbox realizes single gene deletions at an efficiency of 50-100% in seven days. Furthermore, the chromosomal gene replacement of Lp_0537 using a P23 -pyruvate decarboxylase (pdc) expression cassette is accomplished with an efficiency of 35.7%. This study establises a RecE/T-assisted CRISPR genome editing toolbox for L. plantarum WCFS1 and L. brevis ATCC367 and also demonstrate that RecE/T-assisted CRISPR-Cas9 is an effective genome editing system, which can be readily implemented in Lactobacilli.

Aqueous Two-Phase Systems at Large Scale: Challenges and Opportunities

Abstract: Aqueous two-phase systems (ATPS) have proved to be an efficient and integrative operation to enhance recovery of industrially relevant bioproducts. After ATPS discovery, a variety of works have been published regarding their scaling from 10 to 1000 L. Although ATPS have achieved high recovery and purity yields, there is still a gap between their bench-scale use and potential industrial applications. In this context, this review paper critically analyzes ATPS scale-up strategies to enhance the potential industrial adoption. In particular, large-scale operation considerations, different phase separation procedures, the available optimization techniques (univariate, response surface methodology, and genetic algorithms) to maximize recovery and purity and economic modeling to predict large-scale costs, are discussed. ATPS intensification to increase the amount of sample to process at each system, developing recycling strategies and creating highly efficient predictive models, are still areas of great significance that can be further exploited with the use of high-throughput techniques. Moreover, the development of novel ATPS can maximize their specificity increasing the possibilities for the future industry adoption of ATPS. This review work attempts to present the areas of opportunity to increase ATPS attractiveness at industrial levels.

Systems Metabolic Engineering Meets Machine Learning: A New Era for Data-Driven Metabolic Engineering.

Abstract: The recent increase in high-throughput capacity of 'omics datasets combined with advances and interest in machine learning (ML) have created great opportunities for systems metabolic engineering. In this regard, data-driven modeling methods have become increasingly valuable to metabolic strain design. In this review, the nature of 'omics is discussed and a broad introduction to the ML algorithms combining these datasets into predictive models of metabolism and metabolic rewiring is provided. Next, this review highlights recent work in the literature that utilizes such data-driven methods to inform various metabolic engineering efforts for different classes of application including product maximization, understanding and profiling phenotypes, de novo metabolic pathway design, and creation of robust system-scale models for biotechnology. Overall, this review aims to highlight the potential and promise of using ML algorithms with metabolic engineering and systems biology related datasets.

Light-Dependent and Aeration-Independent Gram-Scale Hydroxylation of Cyclohexane to Cyclohexanol by CYP450 Harboring Synechocystis sp. PCC 6803.

Abstract: Oxygenase-containing cyanobacteria constitute promising whole-cell biocatalysts for oxyfunctionalization reactions. Photosynthetic water oxidation thereby delivers the required cosubstrates, that is activated reduction equivalents and O2 , sustainably. A recombinant Synechocystis sp. PCC 6803 strain showing unprecedentedly high photosynthesis-driven oxyfunctionalization activities is developed, and its technical applicability is evaluated. The cells functionally synthesize a heterologous cytochrome P450 monooxygenase enabling cyclohexane hydroxylation. The biocatalyst-specific reaction rate is found to be light-dependent, reaching 26.3 ± 0.6 U gCDW-1 (U = μmol min-1 and cell dry weight [CDW]) at a light intensity of 150 µmolphotons m-2 s-1 . In situ substrate supply via a two-liquid phase system increases the initial specific activity to 39.2 ± 0.7 U gCDW-1 and stabilizes the biotransformation by preventing cell toxification. This results in a tenfold increased specific product yield of 4.5 gcyclohexanol gCDW-1 as compared to the single aqueous phase system. Subsequently, the biotransformation is scaled from a shake flask to a 3 L stirred-tank photobioreactor setup. In situ O2 generation via photosynthetic water oxidation allows a nonaerated process operation, thus circumventing substrate evaporation as the most critical factor limiting the process performance and stability. This study for the first time exemplifies the technical applicability of cyanobacteria for aeration-independent light-driven oxyfunctionalization reactions involving highly toxic and volatile substrates.

Perfusion Cell Culture Decreases Process and Product Heterogeneity in a Head-to-Head Comparison With Fed-Batch.

Abstract: In this study, the authors compared the impacts of fed-batch and perfusion platforms on process and product attributes for IgG1- and IgG4-producing cell lines. A "plug-and-play" approach is applied to both platforms at bench scale, using commercially available basal and feed media, a standard feed strategy for fed-batch and ATF filtration for perfusion. Product concentration in fed-batch is 2.5 times greater than perfusion, while average productivity in perfusion is 7.5 times greater than fed-batch. PCA reveals more variability in the cell environment and metabolism during the fed-batch run. LDH measurements show that exposure of product to cell lysate is 7-10 times greater in fed-batch. Product analysis shows larger abundances of neutral species in perfusion, likely due to decreased bioreactor residence times and extracellular exposure. The IgG1 perfusion product also has higher purity and lower half-antibody. Glycosylation is similar across both culture modes. The first perfusion harvest slice for both product types shows different glycosylation than subsequent harvests, suggesting that product quality lags behind metabolism. In conclusion, process and product data indicate that intra-lot heterogeneity is decreased in perfusion cultures. Additional data and discussion is required to understand the developmental, clinical and commercial implications, and in what situations increased uniformity would be beneficial.

Endotoxin-Free E. coli-Based Cell-Free Protein Synthesis: Pre-Expression Endotoxin Removal Approaches for on-Demand Cancer Therapeutic Production.

Abstract: Approximately one third of protein therapeutics are produced in Escherichia coli, targeting a wide variety of diseases However, due to immune recognition of endotoxin (a lipid component in the E coli cell membrane), these protein products must be extensively purified before application to avoid adverse reactions such as septic shock E coli-based cell-free protein synthesis (CFPS), which has emerged as a promising platform for the development and production of enhanced protein therapeutics, provides a unique opportunity to remove endotoxins prior to protein expression due to its open environment and the absence of live cells Pre-expression endotoxin removal from CFPS reagents could simplify downstream processing, potentially enabling on-demand production of unique protein therapeutics Herein, three strategies for removing endotoxins from E coli cell lysate are evaluated: Triton X-114 two-phase extraction, polylysine affinity chromatography, and extract preparation from genetically engineered, endotoxin-free ClearColi cells It is demonstrated that current protocols for endotoxin removal treatments insufficiently reduce endotoxin and significantly reduce protein synthesis yields Further, the first adaptation of ClearColi cells to prepare cell-free extract with high protein synthesis capability is demonstrated Finally, production of the acute lymphoblastic leukemia therapeutic crisantaspase from reduced-endotoxin extract and endotoxin-free ClearColi extract is demonstrated

Metabolic Engineering of Corynebacterium glutamicum for High‐Level Ectoine Production: Design, Combinatorial Assembly, and Implementation of a Transcriptionally Balanced Heterologous Ectoine Pathway

Abstract: Ectoine is formed in various bacteria as cell protectant against all kinds of stress. Its preservative and protective effects have enabled various applications in medicine, cosmetics, and biotechnology, and ectoine therefore has high commercial value. Industrially, ectoine is produced in a complex high-salt process, which imposes constraints on the costs, design, and durability of the fermentation system. Here, Corynebacterium glutamicum is upgraded for the heterologous production of ectoine from sugar and molasses. To overcome previous limitations, the ectoine pathway taken from Pseudomonas stutzeri is engineered using transcriptional balancing. An expression library with 185,193 variants is created, randomly combining 19 synthetic promoters and three linker elements. Strain screening discovers several high-titer mutants with an improvement of almost fivefold over the initial strain. High production thereby particularly relies on a specifically balanced ectoine pathway. In an optimized fermentation process, the new top producer C. glutamicum ectABCopt achieves an ectoine titer of 65 g L-1 and a specific productivity of 120 mg g-1 h-1 . This process is the first reported example of a simple fermentation process under low-salt conditions using well-established feedstocks to produce ectoine with industrial efficiency. There is a compelling case for more intensive implementation of transcriptional balancing in future metabolic engineering of C. glutamicum.

Dissolvable Microcarriers Allow Scalable Expansion And Harvesting Of Human Induced Pluripotent Stem Cells Under Xeno-Free Conditions

Abstract: The development of bioprocesses capable of producing large numbers of human induced pluripotent stem cells (hiPSC) in a robust and safe manner is critical for the application of these cells in biotechnological and medical applications. Scalable expansion of hiPSC is often performed using polystyrene microcarriers, which have to be removed from the cell suspension using a separation step that causes loss of viable cells. In this study, application of novel xeno-free dissolvable microcarriers (DM) for an efficient and integrated expansion and harvesting of hiPSC is demonstrated. After an initial screening under static conditions, hiPSC culture using DM is performed in dynamic culture, using spinner-flasks. A maximum 4.0 ± 0.8-fold expansion is achieved after 5 days of culture. These results are validated with a second cell line and the culture is successfully adapted to fully xeno-free conditions. Afterwards, cell recovery is made within the spinner flask, being obtained a 92 ± 4% harvesting yield, which is significantly higher than the one obtained for the conventional filtration-based method (45 ± 3%). Importantly, the expanded and harvested hiPSC maintain their pluripotency and multilineage differentiation potential. The results here described represent a significant improvement of the downstream processing after microcarrier-based hiPSC expansion, leading to a more cost-effective and efficient bioprocess.

Exosomes for Non-Invasive Cancer Monitoring.

Abstract: Exosomes, membrane-bound phospholipid vesicles having diameters of 50-200 nm, are secreted by all cell types and circulate in human body fluids. These vesicles are known to carry cellular constituents that are specific to the originating cells (e.g., cytoplasmic/membrane proteins, RNA, and DNA). Thus, exosomes, which are both structurally stable and abundant, are robust indicators of cancers and, as a result, they have been utilized to monitor this disease in a manner that is less invasive than gold standard tissue biopsies. In this review, the history of exosomes and the specific biomarkers present in exosomes that enable accurate monitoring of various diseases are described. In addition, methods for analysis of exosomes and identification of biomarkers are presented with special emphasis being given to isolation and signaling strategies. Lastly, integrated, microfluidic systems developed for exosome-based cancer diagnosis are described and future directions that research in this area will likely take are presented.

Recent Progress in Polyhydroxyalkanoates-Based Copolymers for Biomedical Applications.

Abstract: In recent years, naturally biodegradable polyhydroxyalkanoate (PHA) monopolymers have become focus of public attentions due to their good biocompatibility. However, due to its poor mechanical properties, high production costs, and limited functionality, its applications in materials, energy, and biomedical applications are greatly limited. In recent years, researchers have found that PHA copolymers have better thermal properties, mechanical processability, and physicochemical properties relative to their homopolymers. This review summarizes the synthesis of PHA copolymers by the latest biosynthetic and chemical modification methods. The modified PHA copolymer could greatly reduce the production cost with elevated mechanical or physicochemical properties, which can further meet the practical needs of various fields. This review further summarizes the broad applications of modified PHA copolymers in biomedical applications, which might shred lights on their commercial applications.

Next-Generation Industrial Biotechnology-Transforming the Current Industrial Biotechnology into Competitive Processes.

Abstract: The chemical industry has made a contribution to modern society by providing cost-competitive products for our daily use. However, it now faces a serious challenge regarding environmental pollutions and greenhouse gas emission. With the rapid development of molecular biology, biochemistry, and synthetic biology, industrial biotechnology has evolved to become more efficient for production of chemicals and materials. However, in contrast to chemical industries, current industrial biotechnology (CIB) is still not competitive for production of chemicals, materials, and biofuels due to their low efficiency and complicated sterilization processes as well as high-energy consumption. It must be further developed into "next-generation industrial biotechnology" (NGIB), which is low-cost mixed substrates based on less freshwater consumption, energy-saving, and long-lasting open continuous intelligent processing, overcoming the shortcomings of CIB and transforming the CIB into competitive processes. Contamination-resistant microorganism as chassis is the key to a successful NGIB, which requires resistance to microbial or phage contaminations, and available tools and methods for metabolic or synthetic biology engineering. This review proposes a list of contamination-resistant bacteria and takes Halomonas spp. as an example for the production of a variety of products, including polyhydroxyalkanoates under open- and continuous-processing conditions proposed for NGIB.

Recommendations for Comparison of Productivity Between Fed-Batch and Perfusion Processes

Abstract: Due to the growing interest in integrated continuous processing in the biopharmaceutical industry, productivity comparison of batch-based and continuous processes is considered a challenge. Integrated continuous manufacturing of biopharmaceuticals requires scientists and engineers to collaborate effectively. Differing definitions, for example, of volumetric productivity, may cause confusion in this interdisciplinary field. Therefore, the aim of this communication is to reiterate the standard definitions and their underlying assumptions. Applying them to an exemplary model scenario allows to demonstrate the differences and to develop recommendations for the comparison of productivity of different upstream processes.

Bioenergy and Biorefinery: Feedstock, Biotechnological Conversion, and Products.

Abstract: Biorefinery has been suggested to provide relevant substitutes to a number of fossil products. Feedstocks and conversion technologies have, however, been the bottleneck to the realization of this concept. Herein, feedstocks and bioconversion technologies under biorefinery have been reviewed. Over the last decade, research has shown possibilities of generating tens of new products but only few industrial implementations. This is partly associated with low production yields and poor cost-competitiveness. This review addresses the technical barriers associated with the conversion of emerging feedstocks into chemicals and bioenergy platforms and summarizes the developed biotechnological approaches including advances in metabolic engineering. This summary further suggests possible future advances that would expand the portfolio of biorefinery and speed up the realization of biofuels and biochemicals.

Synthetic Biology Toolkits for Metabolic Engineering of Cyanobacteria.

Abstract: Cyanobacteria are of great importance to Earth's ecology. Due to their capability in photosynthesis and C1 metabolism, they are ideal microbial chassis that can be engineered for direct conversion of carbon dioxide and solar energy into biofuels and biochemicals. Facilitated by the elucidation of the basic biology of the photoautotrophic microbes and rapid advances in synthetic biology, genetic toolkits have been developed to enable implementation of nonnatural functionalities in engineered cyanobacteria. Hence, cyanobacteria are fast becoming an emerging platform in synthetic biology and metabolic engineering. Herein, the progress made in the synthetic biology toolkits for cyanobacteria and their utilization for transforming cyanobacteria into microbial cell factories for sustainable production of biofuels and biochemicals is outlined. Current techniques in heterologous gene expression, strategies in genome editing, and development of programmable regulatory parts and modules for engineering cyanobacteria towards biochemical production are discussed and prospected. As cyanobacteria synthetic biology is still in its infancy, apart from the achievements made, the difficulties and challenges in applying and developing genetic toolkits in cyanobacteria for biochemical production are also evaluated.

Structural features on the substrate-binding surface of fungal lytic polysaccharide monooxygenases determine their oxidative regioselectivity

Abstract: Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave many of nature's most recalcitrant polysaccharides by acting on the C1- and/or C4-carbon of the glycosidic bond. Here, the results of an extensive mutagenesis study on three LPMO representatives, Phanerochaete chrysosporium LPMO9D (C1-oxidizer), Neurospora crassa LPMO9C (C4), and Hypocrea jecorina LPMO9A (C1/C4), are reported. Using a previously published indicator diagram, the authors demonstrate that several structural determinants of LPMOs play an important role in their oxidative regioselectivity. N-glycan removal and alterations of the aromatic residues on the substrate-binding surface are shown to alter C1/C4-oxidation ratios. Removing the carbohydrate binding module (CBM) is found not to alter the regioselectivity of HjLPMO9A, although the effect of mutational changes is shown to increase in a CBM-free context. The accessibility to the solvent-exposed axial position of the copper-site reveales not to be a major regioselectivity indicator, at least not in PcLPMO9D. Interestingly, a HjLPMO9A variant lacking two surface exposed aromatic residues combines decreased binding capacity with a 22% increase in synergetic efficiency. Similarly to recent LPMO10 findings, our results suggest a complex matrix of surface-interactions that enables LPMO9s not only to bind their substrate, but also to accurately direct their oxidative force.

Gated Materials: Installing Macrocyclic Arenes-Based Supramolecular Nanovalves on Porous Nanomaterials for Controlled Cargo Release.

Abstract: Supramolecular nanovalves are an emerging class of important elements that are functionalized on the surfaces of inorganic or hybrid nanocarriers in the constructions of smart cargo delivery systems. Taking advantage of the pseudorotaxane structure via host-guest complexation and the dynamic nature of supramolecular interactions, macrocyclic arene-based supramolecular nanovalves have shown great promise in the applications of drug delivery and controlled release. Careful selection of diverse external stimuli, such as pH variations, temperature changes, redox, enzymes, light irradiation, and competitive binding, can activate the opening and closing of the nanovalves by altering the supramolecular structure or binding affinities. Meanwhile, the porous solid supports in controlled release systems also play an important role in the functionalities of the nanocarriers, which include, but not limited to, mesoporous silica nanoparticles (MSNs), metal-organic frameworks (MOFs), core-shell nanomaterials, and rare-earth porous nanomaterials. The elaborate decoration by macrocyclic arenes-based supramolecular nanovalves on porous nanomaterials has provided intelligent controlled release platforms. In this review, we will focus on the overview of supramolecular nanovalves based on two typical macrocyclic arenes, that is, calixarenes and pillarenes, and their operation manners in the controlled release processes.

Evolutionary engineering of Corynebacterium glutamicum.

Abstract: A unique feature of biotechnology is that we can harness the power of evolution to improve process performance. Rational engineering of microbial strains has led to the establishment of a variety of successful bioprocesses, but it is hampered by the overwhelming complexity of biological systems. Evolutionary engineering represents a straightforward approach for fitness-linked phenotypes (e.g., growth or stress tolerance) and is successfully applied to select for strains with improved properties for particular industrial applications. In recent years, synthetic evolution strategies have enabled selection for increased small molecule production by linking metabolic productivity to growth as a selectable trait. This review summarizes the evolutionary engineering strategies performed with the industrial platform organism Corynebacterium glutamicum. An increasing number of recent studies highlight the potential of adaptive laboratory evolution (ALE) to improve growth or stress resistance, implement the utilization of alternative carbon sources, or improve small molecule production. Advances in next-generation sequencing and automation technologies will foster the application of ALE strategies to streamline microbial strains for bioproduction and enhance our understanding of biological systems.

Engineered Skin Tissue Equivalents for Product Evaluation and Therapeutic Applications.

Abstract: The current status of skin tissue equivalents that have emerged as relevant tools in commercial and therapeutic product development applications is reviewed. Due to the rise of animal welfare concerns, numerous companies have designed skin model alternatives to assess the efficacy of pharmaceutical, skincare, and cosmetic products in an in vitro setting, decreasing the dependency on such methods. Skin models have also made an impact in determining the root causes of skin diseases. When designing a skin model, there are various chemical and physical considerations that need to be considered to produce a biomimetic design. This includes designing a structure that mimics the structural characteristics and mechanical strength needed for tribological property measurement and toxicological testing. Recently, various commercial products have made significant progress towards achieving a native skin alternative. Further research involve the development of a functional bilayered model that mimics the constituent properties of the native epidermis and dermis. In this article, the skin models are divided into three categories: in vitro epidermal skin equivalents, in vitro full-thickness skin equivalents, and clinical skin equivalents. A description of skin model characteristics, testing methods, applications, and potential improvements is presented.