Recent noteworthy advances in the development of high-performing microbial and mammalian strains have enabled the sustainable production of bio-economically valuable substances such as bio-compounds, biofuels, and biopharmaceuticals. However, to obtain an industrially viable mass-production scheme, much time and effort are required. The robust and rational design of fermentation processes requires analysis and optimization of different extracellular conditions and medium components, which have a massive effect on growth and productivity. In this regard, knowledge- and data-driven modeling methods have received much attention. Constraint-based modeling (CBM) is a knowledge-driven mathematical approach that has been widely used in fermentation analysis and optimization due to its capabilities of predicting the cellular phenotype from genotype through high-throughput means. On the other hand, machine learning (ML) is a data-driven statistical method that identifies the data patterns within sophisticated biological systems and processes, where there is inadequate knowledge to represent underlying mechanisms. Furthermore, ML models are becoming a viable complement to constraint-based models in a reciprocal manner when one is used as a pre-step of another. As a result, more predictable models are produced. This review highlights the applications of CBM and ML independently and the combination of these two approaches for analyzing and optimizing fermentation parameters.

In the attempt to bridge the widening gap from DNA sequence to biological function, we developed a novel methodology to assemble Long-Adapter Single-Strand Oligonucleotide (LASSO) probe libraries that enabled the massively multiplexed capture of kilobase-sized DNA fragments for downstream long read DNA sequencing or expression. This method uses short DNA oligonucleotides (pre-LASSO probes) and a plasmid vector that supplies the backbone for the mature LASSO probe through Cre-Loxp intramolecular recombination. This strategy generates high quality LASSO probes libraries (~46% of probes). We performed NGS analysis of the post-capture PCR amplification of DNA circles obtained from the LASSO capture of 3087 E.coli ORFs spanning from 400- to 4,000 bp. The median enrichment of all targeted ORFs versus untargeted ORFs was 30 times. For ORFs up to 1kb in size, targeted ORFs were enriched up to a median of 260-fold. Here, we show that LASSO probes obtained in this manner, are able to capture full-length open reading frames from total human cDNA. Furthermore, we show that the LASSO capture specificity and sensitivity is sufficient for target capture from total human genomic DNA template. This technology can be used for the preparation of long-read sequencing libraries and for massively multiplexed cloning of human sequences.

Viral surrogates to screen for virus inactivation (VI) can be a faster, cheaper and safer alternative to third-party testing of pathogenic BSL2 (Biosafety Level 2) model viruses. Although the bacteriophage surrogate, Ø6, has been used to assess low pH BSL2 VI, it has not been used for evaluation of detergent-mediated VI. Furthermore, Ø6 is typically assayed through host cell infectivity which introduces the risk of cross-contaminating other cell lines in the facility. To circumvent contamination, we developed an in-house RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) assay for selective detection of active Ø6 from a population of live and dead phage. The RT-qPCR assay was used to evaluate Ø6 inactivation in cell culture fluid of monoclonal antibody and fusion protein. Complementary Ø6 infectivity was also conducted at a third-party testing facility. The Ø6 RT-qPCR and infectivity data was modeled against VI of three BSL2 viruses, X- MuLV, A- MuLV and HSV-1 in corresponding therapeutics. Both Ø6 methods demonstrate that any VI agent showing Ø6 clearance of ≥ 2.5 logs would demonstrate complete BSL2 VI of ≥ 4.0 logs. Compared to BSL2 virus testing, this in-house Ø6 RT-qPCR tool can screen VI agents at 5% the cost and a turnaround time of 2-3 days versus 4-7 months.

Because of their excellent therapeutic potential, mesenchymal stem cells (MSCs) have been used as cell therapeutics for various diseases. However, the survival rate and duration of MSCs after transplantation are extremely low and short, respectively. To solve these problems, in this study, we prepared multicellular spheroids of MSCs and investigated their survival and function after intravenous injection in mice. The murine adipose-derived MSC line m17.ASC was cultured in agarose-based microwell plates to obtain size-controlled m17.ASC spheroids of an average diameter and cell number of approximately 170 μm and 1100 cells/spheroid, respectively. The intravenously injected m17.ASC spheroids mainly accumulated in the lung and showed a higher survival rate than suspended m17.ASC cells during the experimental period of 7 days. m17.ASC spheroids efficiently reduced the lipopolysaccharide-induced increase in plasma concentrations of interleukin-6 and tumor necrosis factor-α. These results indicate that spheroid formation improved the pulmonary delivery and survival of MSCs, as well as their therapeutic potential against inflammatory pulmonary diseases.

We herein describe an ingenious centrifugal microfluidic system to accomplish a fully automated serial dilution. The liquid flow on the disc was automatically regulated by utilizing ferrowax microvalves systematically integrated into the channels within the specially designed metering structure. By opening the differently positioned microvalves through irradiation of IR laser to allow metering, the same amount of diluent was serially eluted to the dilution chamber from the same diluent chamber. After dilution, the diluted samples were automatically delivered to the respective final product chambers by appropriately opening or closing the microvalves in the connecting channels, followed by rotating the disc. Based on this unique design principle, six consecutive two-fold and ten-fold dilutions were successfully achieved, yielding excellent accuracy in a wide dynamic range up to six orders of magnitude. Very importantly, the overall serial dilution process, including the diluent addition, mixing, and product transfer steps, was completed very rapidly within five minutes, due to the minimized procedures enabled by the automated actuation of the ferrowax microvalves at the rationally designed positions. The centrifugal microfluidic system would serve as a powerful elemental tool to realize the fully automated diagnostic microsystem involving the serial dilution process.

Difference between two strong cation-exchange resins, namely sulfonium type and sulfate type regarding both their salt tolerance and hydrophobicity were investigated. There is only tiny variation between sulfate and sulfonic group and at the first glance it seems unlikely that it could be the reason for changed selectivity and salt tolerance that was detected in our preliminary experiments. For that reason salt tolerance and hydrophobicity of both ligands was investigated by using two representative polymethacrylate-based ion exchangers as for the sulfonium type TOYOPEARL GigaCap S-650M and for the sulfate type TOYOPEARL Sulfate-650F. In addition some in-silico calculations were performed for model substances representing the sulfonium and sulfate group, and significant differences were calculated regarding their hydrophobicity. These experiments confirmed the working hypothesis that salt tolerance and higher affinity and selectivity for some human plasma derived vitamin K dependent clotting factors and inhibitors are interrelated and dependent from the presence of the sulfate group. The affinity for these proteins was experimentally verified by separation of clotting factor IX from the prothrombin complex concentrate. Presented results show that a simple and fast separation between clotting factor IX and other vitamin K dependent clotting factors II, VII and X is possible, only if the resin with the sulfate, and not with sulfonic acid ligand was applied. Consequently, an immediate application of undiluted feedstock or the eluate from previous isolation step to sulfate resin is possible, and a significant optimization of downstream process can be achieved.

Current production costs of microalgal biomass indicate that only highly-productive cultivation facilities will approach commercial feasibility. Geographical site selection for siting those facilities is critical for achieving target productivities. The aim of this study was to provide a semi-empirical estimation of microalgal biomass and lipids productivity in South America. Simulated-climate was programed in environmental photobioreactors (PhenometricsTM) for a simulation of cultivation in open raceway ponds at different geographical sites. The mean annual South American biomass productivity of 20-cm deep ponds was 12 ± 4 g · m- 2 · d-1. The most productive regions were clustered in the subtropical and tropical regions of the continent. Fortaleza (Brazil) showed a low seasonality and a high annual mean productivity of 23 g · m-2 · d-1 in 5-cm deep ponds. Lipids accumulation and productivity in Fortaleza showed a high microalgal oil accumulation up to 46% (w/w) and a maximum oil productivity of 5 g · m-2 · d-1 for biomass containing around 20% lipids (w/w). This study provides the first semi-empirical estimation of microalgal productivity in South America and supports a high potential of a vast region of the continent.

Microfluidic devices have been introduced for phenotypic screening of zebrafish larvae in both fundamental and pre-clinical research. One of the remaining challenges for the broad use of microfluidic devices is their limited throughput, especially in behavioural assays. Previously, we introduced the tail locomotion of a semi-mobile zebrafish larva evoked on-demand with electric signal in a microfluidic device. Here, we report the lessons learned for increasing the number of specimens from one to four larvae in this device. Multiple parameters including loading and testing time per fish and loading and orientation efficiencies were refined to optimize the performance of modified designs. Simulations of the flow and electric field within the final device provided insight into the flow behavior and functionality of traps when compared to previous single-larva devices. Outcomes led to a new design which decreased the testing time per larva by approximately 60%. Further, loading and orientation efficiencies increased by more than 80%. Critical behavioural parameters such as response duration and tail beat frequency were similar in both single and quadruple-fish devices. The optimized microfluidic device has significant advantages for greater throughput and efficiency when behavioral phenotyping is required in various applications, including chemical testing in toxicology and gene screening.

Given the potential applications of gas vesicles (GVs) in multiple fields including antigen-displaying and imaging, heterologous reconstitution of synthetic GVs is an attractive and interesting study that has translational potential. Here, we attempted to express and assemble GV proteins (GVPs) into GVs using the model eukaryotic organism Saccharomyces cerevisiae. We first selected and expressed two core structural proteins, GvpA and GvpC from cyanobacteria Anabaena flos-aquae and Planktothrix rubescens, respectively. We then optimized the protein expression conditions and validated GV assembly in the context of cell flotation and GV shapes. We found that when two copies of AnaA were integrated into the genome, it resulted in cell floatation and GV production regardless of GvpC expression. Next, we co-expressed chaperone-RFP with the GFP-AnaA to aid the AnaA aggregation. The co-expression of individual chaperones (Hsp42, Sis1, Hsp104, and GvpN) with AnaA led to the formation of larger inclusions and enhanced the sequestration of AnaA into the perivacuolar site. To our knowledge, this represents the first study on reconstitution of GVs in S. cerevisiae. Our results could provide insights into optimizing conditions for heterologous protein expressions as well as the reconstitution of other synthetic microcompartments in yeast.

Failure in the prevention of cross-transmission from contaminated gloves has been recognized as an important factor that contributes to the spread of several healthcare-associated infections. Ex situ coating process with silver nanoparticles (AgNPs) using Eucalyptus citriodora ethanolic leaf extract as reducing and capping agents to coat glove surfaces has been developed to prevent this mode of transmission. Elemental analysis of coated gloves showed 24.8 Wt% silver densely adhere on the glove surface. The coated gloves fully eradicated important hospital-acquired pathogens including Gram-positive bacteria, Gram-negative bacteria, and yeasts within 1 h. The coated gloves showed significant reduction, an average of 5 logs when tested against all standard strains and most clinical isolates (p < 0.01). Following prolonged exposure, the coating significantly reduced the numbers of most adhered pathogenic species, compared with uncoated gloves (p < 0.0001), which was observed by fluorescence microscopy. Scanning electron microscopy further confirmed that AgNPs coated-gloves reduced microbial adhesion of mixed-species biofilms, compared with uncoated gloves. A series of contamination and transmission assays demonstrated no transmission of viable organisms. Biocompatibility analysis confirmed high cell viability of HaCaT and L929 cells at all concentrations of AgNPs tested. The coated gloves were non-toxic with direct contact with L929 cells.

The European corn borer (ECB) Ostrinia nubilalis is a widespread pest of cereals. Mating disruption with the sex pheromone is a potentially attractive method for managing this pest. The goal of this study was to develop a biotechnological method for the production of ECB sex pheromone. Our approach was to engineer the oleaginous yeast Yarrowia lipolytica to produce (Z)-11-tetradecenol (Z11-14:OH), which can be chemically acetylated to (Z)-11-tetradecenyl acetate (Z11-14:OAc), the main pheromone component of the Z-race of O. nubilalis. Fatty acyl-CoA desaturases (FAD) and fatty acyl-CoA reductases (FAR) from nine different species of Lepidoptera were screened individually and in combinations. A titer of 29.2±1.6 mg/L Z11-14:OH was reached in small-scale cultivation with an optimal combination of a FAD (Lbo_PPTQ) from Lobesia botrana and FAR (HarFAR) from Helicoverpa armigera. When the second copies of FAD and FAR genes were introduced, the titer improved 2.1-fold. The native FAS1 gene’s overexpression led to a further 1.5-fold titer increase. When the same engineered strain was cultivated in controlled 1 L bioreactors in fed-batch mode, 188.1±13.4 mg/L of Z11-14:OH was obtained. Fatty alcohols were chemically acetylated to obtain Z11-14:OAc. Electroantennogram experiments showed that males of the Z-race of O. nubilalis were responsive to biologically-derived pheromone blend. Behavioral bioassays in a wind tunnel revealed attraction of male O. nubilalis at a level similar to that of the chemically synthesized pheromone used as a control, although full precopulatory behavior was observed less often. The study paves the way for the production of ECB pheromone by fermentation.

Ursolic acid (UA) is a ursane-type pentacyclic triterpenoid compound, naturally produced in plants via specialized metabolism and exhibits vast range of remarkable physiological activities and pharmacological manifestations. Owing to significant safety and efficacy in different medical conditions, UA may serve as a backbone to produce its derivatives with novel therapeutic functions. This review systematically provides an overview of the pharmacological activities, acquisition methods and structural modification methods of UA. In addition, we focused on the synthetic modifications of UA to yield its valuable derivatives with enhanced therapeutic potential. Furthermore, harnessing the essential advances for green synthesis of UA and its derivatives by advent of metabolic engineering and synthetic biology are highlighted. In combination with the advantages of UA biosynthesis and transformation strategy, large-scale production and applications of UA is a promising platform for further exploration.

Western equine encephalitis virus (WEEV) can cause lethal encephalitis in humans and equines and represents a serious public health threat in many countries. Therefore, development of efficient vaccines against WEEV remains an important challenge in the field of disease control. This study described for the first time successful production of WEEV virus-like particles (VLPs) in insect cells using recombinant baculoviruses. This well-established expression system is very suitable for production of WEEV VLPs. The immune experiment herein in mice showed that the VLPs formulated with 206-adjuvant were responsible for the stronger-VLP-specific cellular immune response, and were able to induce the secretion of IL-2, IL-4, IFN-γ and production of high titer antibodies that can effectively neutralize the WEEV pseudoviruses. The WEEV VLPs from insect cells could provide a new, safe, non-replicating and effective vaccine candidate against WEEV infections.

Bioprocess development and optimization is a challenging, costly, and time-consuming effort. In this multidisciplinary task, upstream processing (USP) and downstream processing (DSP) are conventionally considered distinct disciplines. This consideration fosters “one-way” optimization without considering interdependencies between unit operations; thus, the full potential of the process chain cannot be achieved. Therefore, it is necessary to fully integrate USP and DSP process development to provide balanced biotechnological production processes. The aim of the present study was to investigate how different host/leader/antigen binding fragment (Fab) combinations in E. coli expression systems influence USP and primary recovery performance and the final product quality. We ran identical fed-batch cultivations with 16 different expression clones to study growth and product formation kinetics, as well as centrifugation efficiency, viscosity, extracellular DNA, and endotoxin content, which are important parameters in DSP. We observed a severe influence on cell growth, product titer, extracellular product, and cell lysis, accompanied by a significant impact on the analyzed parameters of DSP performance. Our results provide the basis for establishing integrated process development considering interdependencies between USP and DSP. These interdependencies need to be understood for rational decision-making and efficient process development.

In recent years Next-Generation Sequencing (NGS) based methods to detect mutations in biotherapeutic transgene products have become a key quality step deployed during the development of manufacturing cell line clones. Previously we reported on a higher throughput, rapid mutation detection method based on amplicon sequencing (targeting transgene RNA) and detailed its implementation to facilitate cell line clone selection. By gaining experience with our assay in a diverse set of cell line development programs, we improved the computational analysis as well as experimental protocols. Here we report on these improvements as well as on a comprehensive benchmarking of our assay. We evaluated assay performance by mixing amplicon samples of a verified mutated antibody clone with a non-mutated antibody clone to generate spike-in mutations from ~60% down to ~0.3% frequencies. We subsequently tested the effect of 16 different sample and NGS library preparation protocols on the assay’s ability to quantify mutations and on the occurrence of false-positive background error mutations (artifacts). Our evaluation confirmed assay robustness, established a high confidence limit of detection of ~0.6%, and identified protocols that reduce error levels thereby significantly reducing a source of false positives that bottlenecked the identification of low-level true mutations.

The production of recombinant proteins usually reduces cell fitness and the growth rate of producing cells. The growth disadvantage favors faster-growing non-producer mutants. Therefore, continuous bioprocessing is hardly feasible in Escherichia coli due to the high escape rate. We investigated the stability of E. coli expression systems under long-term production conditions and how metabolic load triggered by recombinant gene expression influences the characteristics of mutations. We conducted iterated fed-batch-like microbioreactor cultivations under production conditions. We used the easy-to-produce green fluorescent protein (GFP) and a challenging antigen-binding fragment (Fab) as model proteins, and BL21(DE3) and BL21Q strains as expression hosts. In comparative whole genome sequencing analyses, we identified mutations that allowed cells to grow unhindered despite recombinant protein production. A T7 RNA polymerase expression system is only conditionally suitable for long-term cultivation under production conditions. Mutations leading to non-producers occur in either the T7 RNA polymerase gene or the T7 promoter. The host RNA polymerase-based BL21Q expression system remained stable in the production of GFP in long-term cultivations. For the production of Fab, mutations in lacI of the BL21Q derivatives had positive effects on long-term stability. Our results indicate that adaptive evolution carried out with genome-integrated E. coli expression systems in microtiter cultivations under industrial relevant production conditions is an efficient strain development tool for production hosts.

We report a novel Cultured Neuronal Network on a chip as a viable alternative to current culture methods for the analysis of neuronal network formation and evolution of the structural properties of the network's graph . This innovative microfluidic chip fabricated from Polydimethylsiloxane, vinyl, and glass offers worthy features such as the posibility to develop and produce custom designs effortlessly, easy handling and monitoring, culture medium renewal, and reducing the exposure to contamination. Due to these benefits, longer survival of the neuronal networks are observed in comparison to conventional methods. In addition to the fast and cost-effective manufacturing of the chip, this technology provides a significant step forward in the studies of neuronal network development and many challening applications such as drug testing of in vitro cell culture models .

Biomolecules are increasingly attractive templates for the synthesis of functional nanomaterials. Chief among them are the plant Tobacco mosaic virus (TMV) and Barley stripe mosaic virus (BSMV) due to their high aspect ratio, narrow size distribution, diverse biochemical functionalities presented on the surface, and compatibility with a number of chemical conjugations. These properties are also easily manipulated by genetic modification to enable the synthesis of a range of metallic and non-metallic nanomaterials for diverse applications. This article reviews the characteristics of TMV, BSMV, and their virus-like particle (VLP) derivatives and how these may be manipulated to extend their use and function. A focus of recent efforts has been on greater understanding and control of the self-assembly processes that drive biotemplate formation. We briefly outline how these features have been exploited in engineering applications such as sensing, catalysis, and energy storage, and discuss emerging advances that promise to accelerate the development of these biotemplates for widescale industrial use.