Synthetic biology has promoted the development of biosensors as tools for detecting trace substances. In the past, biosensors based on synthetic biology have been designed on living cells, but the development of cell biosensors has been greatly limited by defects such as the obstruction of cell membrane. However, the advent of cell-free synthetic biology addresses these limitations. Biosensors based on the cell-free protein synthesis system have the advantages of higher safety, higher sensitivity, and faster response time over cell biosensors, which makes cell-free biosensors have a broader application prospect. This review summarizes the workflow of various cell-free biosensors, including the identification of analytes and signal output. The detection range of cell-free biosensors is greatly enlarged by different recognition mechanisms and output methods. In addition, the review also discusses the applications of cell-free biosensors in environmental monitoring and health diagnosis, as well as existing deficiencies and aspects that should be improved. In the future, through continuous improvement and optimization, the potential of cell-free biosensors will be stimulated, and their application fields will be expanded.

Transient gene expression (TGE) using mammalian cells is an extensively used technology for production of antibodies and recombinant proteins and has been widely adapted by both academic and industrial labs. Chinese Hamster Ovary (CHO) cells have become one of the major work horses for TGE of recombinant antibodies due to their attractive features: post-translational modifications, adaptation to high cell densities, and use of serum-free media. In this study, we describe the optimization of parameters for TGE for antibodies from CHO cells. Through a matrix evaluation of multiple factors including inoculum, transfection conditions, amount and type of DNA used and post-transfection culture conditions, we arrived at an optimized process with higher titer and reduced costs and time, thus increasing the overall efficiency of early antibody material supply. We investigated the amount of coding DNA and the influence of size of the transfection complex on the in vitro efficiency of the transfection. Generation of the transfection complex in serum-free medium leads to the prompt formation of an optimal-sized polyplex, and is independent of the relative amount of coding DNA used for a successful transfection outcome.

Self-sufficient cytochromes P450 of the sub-family CYP116B have gained great attention in biotechnology due to their ability to catalyze challenging reactions towards a wide range of organic compounds without the need of a separate reductase partner. However, these P450s are often unstable in solution and their activity is limited to short reaction time. As the isolated heme domain of CYP116B5 has been shown to work as a peroxygenase with H2O2 without the need for expensive NAD(P)H, in this work protein engineering was used to generate a chimeric enzyme (CYP116B5-SOX), in which the native reductase domain is replaced by a monomeric sarcosine oxidase (MSOX) that is able to produce H2O2 with a controlled and continuous release in time. The full-length form enzyme (CYP116B5-fl) is expressed and characterized for the first time, allowing a detailed comparison to both the isolated heme domain (CYP116B5-hd) and CYP116B5-SOX. The catalytic activity of the three forms of the enzyme was studied using p-nitrophenol as substrate, and adding NADPH (CYP116B5-fl), H2O2 (CYP116B5-hd) and sarcosine (CYP116B5-SOX) as direct or indirect source of electrons. CYP116B5-SOX outperforms CYP116B5-fl by 10 folds and CYP116B5-hd by 3 folds, in terms of p-nitrocatechol produced per mg of enzyme per minute. CYP116B5-SOX represents an optimal model to exploit CYP116B5 and the same protein engineering approach could be used for P450s of the same class.

The scale-up of bioprocesses is still one of the major obstacles in biotechnological industry. Scale-down bioreactors were identified as valuable tools to investigate the heterogeneities observed in large-scale tanks in laboratory-scale. Additionally, computational fluid dynamics (CFD) simulations can be used to gain information about fluid flow in tanks used for production. Here we present the rational design and comprehensive characterization of a scale-down setup, in which a flexible and modular plug-flow reactor is connected to a stirred tank bioreactor. With the help of CFD the mixing time difference between differently scaled bioreactors were evaluated and used as scale-down criterium. Additionally, it was used to characterize the setup at conditions were experiments could technically not be performed. This was the first time a scale-down setup was tested on high cell density Escherichia coli cultivations to produce industrial relevant antigen-binding fragments (Fab). Reduced biomass and product yields were observed during the scale-down cultivations. Additionally, the intracellular Fab fraction was increased by using the setup. The results show that including CFD in the design and characterization of a scale-down reactor can help to keep a connection to production scale and also gain intensive knowledge about the setup, which enhances usability.

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.

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.

Genome editing and gene expression engineering using CRISPR-Cas systems in plants usually rely on labor-intensive tissue culture approaches to generate stably transformed plants that express the components of the reaction. Viral vectors have demonstrated to be a quick and effective alternative to express multiple guide RNAs, DNA templates for homologous recombination, and even Cas nucleases. Here we have developed an improved vector system based on tobacco rattle virus (TRV) to simplify logistics in genome editing and gene silencing approaches. The new system consists in a single Agrobacterium tumefaciens clone co-transformed with two compatible mini binary vectors from which TRV RNA1 and an engineered version of TRV RNA2 are expressed. Sequences of recombinant proteins, gene fragments for virus-induced gene silencing (VIGS) or guide RNAs can be easily inserted by one-step digestion-ligation and homology-based cloning methods in the RNA2 plasmid to produce vectors with a size substantially smaller than usual. Using this new one-Agrobacterium TRV mini vector system, we show robust VIGS of an endogenous host gene after infiltration of bacterial suspensions at low optical densities, and efficient production of recombinant proteins in Nicotiana benthamiana. Most importantly, we also show highly efficient heritable genome editing in more than half of the seedling originating from inoculated N. benthamiana plants that express Cas9.

The pandemic outbreaks of coronavirus disease 2019 (COVID-19) was first discovered in Wuhan, Hubei, China in December 2019. The COVID-19 was caused by the novel coronavirus, namely severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It took 30 days to spread to all provinces of China [1]. Recently, the confirmed cases of COVID-19 have been reported from about 200 countries or regions on March 30, 2020, and killed almost 30 thousand people [2]. Efficient identification of the infection by SARS-CoV-2 has been one of the most important tasks to facilitate all the following counter measurements in dealing with infectious disease. In Taiwan, a COVID-19 Open Science Platform adhering to the spirit of open science: sharing sources, data, and methods to promote progress in academic research while corroborating findings from various disciplines has established in mid-February 2020, for collaborative research in support of the development of detection methods, therapeutics, and a vaccine for COVID-19. Research priorities include infection control, epidemiology, clinical characterization and management, detection methods (including viral RNA detection, viral antigen detection, and serum antibody detection), therapeutics (neutralizing antibody and small molecule drugs), vaccines, and SARS-CoV-2 pathogenesis. In addition, research on social ethics and the law are included to take full account of the impact of the COVID-19 virus.

Substituted alkyl pyrazines - other than being extracted from various natural sources such as coffee beans, cocoa beans, nuts and vegetables - can be synthesized by the use of traditional chemical methods or by the help of certain microorganisms. The importance of pyrazines for food industry is expected to grow in the upcoming years due to the higher demand for ready meals, coffee and chocolate drinks; the roasty, nutty and earthy smell is reminiscent for coffee and cocoa depending on substitution and concentration of pyrazines. The growing awareness of people about the ingredients and the origin of their daily food has strongly influenced the market with labels like ’organic‘ and ’natural‘. Many flavor ingredients prepared by biotechnology methods have conquered the market in recent years and are destined to replace and optimize the ineffective (0.01% pyrazine/kg biomass) extraction from plants or animal sources. This overview focuses on the achievements and the upcoming challenges in pyrazine synthesis. Major parts deal with the extraction of natural products from sugar molasses, the chemical synthesis, fermentation by microorganisms and preparative methods by biocatalysis. The different types of production are decisive for the declaration and value of the final product and can span from 200-3500 $/kg for the synthetically produced or the naturally extracted 2,5-dimethylpyrazine, respectively.

It is common practice in the development of bioprocesses to genetically modify a microorganism and study a large number of resulting mutants in order to select the ones that perform best for use at the industrial scale. At industrial scale, strict nutrient-controlled growth conditions are imposed to control the metabolic activity and growth rate of the microorganism, thereby enhancing the expression of the product of interest. Although it is known that microorganisms that perform best under these strictly controlled conditions are not the same as the ones that perform best under uncontrolled batch conditions, screening, and selection is predominantly performed under batch conditions. Tools that afford high throughput on the one hand and dynamic control over cultivation conditions on the other hand are not yet available. Microbioreactors offer the potential to address this problem, resolving the gap between bioprocess development and industrial scale use. In this review, we highlight the current state-of-the-art of microbioreactors that offer the potential to screen microorganisms under dynamically controlled conditions. We classify them into: (i) microtiter plate-based platforms, (ii) microfluidic chamber-based platforms, and (iii) microfluidic droplet-based platforms. We conclude this review by discussing the opportunities of nutrient-fed microbioreactors in the field of biotechnology.

D-Allulose has many health-benefiting properties, physiological functions, and sustainable applications in food, pharmaceutical, and nutrition industries. The aldol reaction based route is a very promising alternative to Izumoring strategy in D-allulose production. Remarkable studies have been reported in this field, but still suffer from by-product formation and costly purified enzyme involvement. In the present study, we explored the glycerol assimilation, alditol oxidase, alcohol dehydrogenase, aldolase, and dephosphorylation pathways, and modularly designed, assembled, and optimized the D-allulose synthetic cascade in Escherichia coli envelop. We achieved an efficient whole-cell catalyst that produces only D-allulose from cheap glycerol feedstock, eliminating the involvement of purified enzymes. Detailed process optimization improved the D-allulose titer by 1500.00%. Finally, the production was validated in 3-L scale using a 5-L fermenter, and 5.67 g/L D-allulose was produced with a molar yield of 31.43%. This study provided a facile approach to produce D-allulose from glycerol feedstock.

The use of adeno-associated viruses (AAV) as vectors for gene and cell therapy has risen considerably in recent years. Consequently, the amount of AAV vectors required during the validation and clinical trials has also increased. AAV serotype 6 (AAV6) is well-documented for its efficiency in transducing different cell types and has been successfully used in gene and cell therapy protocols. However, the number of vectors required to effectively deliver the transgene to one single cell has been estimated at 106 viral genomes (VG). Overall, this means that large-scale production of AAV6 is needed. Suspension cell-based platforms are currently limited to low-cell-density productions, hindering the potential of this production process to increase yields. Here, we investigate the improvement of the production of AAV6 at higher cell densities. The production was performed by transient transfection of HEK293SF cells. When the plasmid DNA is provided on a cell basis, the production can be carried out at medium cell density without effects on cell-specific titer or particle functionality, resulting in titers above 1010 VG/mL. Medium supplementation alleviated the cell density effect, in terms of VG/cell, at high-cell-density productions. On the other hand, the cell-specific functional titer was not maintained, and further studies are necessary to understand the observed limitations. The medium-cell-density production method reported here lays the foundation for large-scale process operations, potentially solving the current vector shortage in AAV manufacturing.

In the past decade, recombinant adeno-associated virus (rAAV) has gained increased attention as a prominent gene therapy technology to treat monogenetic disease. One of the challenges in rAAV production is the enrichment of full-rAAV particles containing the gene of interested (GOI) payload. Herein, we demonstrated that by adjusting the mobile phase properties of anion-exchange chromatography (AEX), Empty and Full separation of rAAV was improved in monolith based preparative AEX chromatography. When compared to the baseline method using NaCl, the presence of tetraethylammonium acetate (TEA-Ac) in the AEX mobile phase resulted in enhanced resolution (from 0.75 to 1.23) between Empty and Full peaks by salt linear gradient elution, as well as increased the percentage of full-rAAV particles from 20% to 36% and GOI genome recovery (from 59% to 62%). Furthermore, a dual wash + step elution AEX method was developed to harness TEA-Ac contribution on Empty and Full separation in the first wash (wash1) step while removing TEA-Ac in the second wash (wash2) step to ensure product safety. The resulting optimized AEX purification method could be easily adapted in scaled-up manufacturing and could also be applied to purification processes involving other AAV serotypes facing similar Empty and Full rAAV separation challenges.

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.

Bioprocess optimization for cell-based therapies is a resource heavy activity. To reduce the associated cost and time, it is advantageous to carry out process development in small volume systems, with the caveat that such systems be predictive for process scaleup. The transport of oxygen from the gas phase into the culture medium, characterized using the volumetric mass transfer coefficient, kLa, has been identified as a critical parameter for predictive process scaleup. In both large- and small-scale bioreactors, kLa is controlled via mixing, with the method employed dependent upon the size of the reactor. However, existing microplate bioreactor platforms, beneficial for their low working volumes and throughput and automation capabilities, struggle to achieve desired kLa for mammalian cell cultures. Here, we describe the development and testing of a 96-well microplate with integrated Redbud Posts to provide mixing and thus enhanced kLa. Mixing characteristics were investigated, with actuating Redbud Posts shown (visually) to increase convective transport while producing enhanced kLa, providing means to mimic macroscale mammalian cell growth conditions at the microscale. Improved cell growth rates with mixing was demonstrated for two cell types, indicating the potential for this technology to play a valuable role in early stage bioprocess development and optimization.

Cas9 nucleases have become the most versatile tool for genome editing projects in a broad range of organisms. The recombinant production of Cas9 nuclease is desirable for in vitro activity assays or the preparation of ribonucleoproteins (RNPs) for DNA-free genome editing approaches. For the rapid production of Cas9, we explored the use of a recently established cell-free lysate from tobacco (Nicotiana tabacum L.) BY-2 cells. Using this system, the 130-kDa Cas9 nuclease from Staphylococcus aureus (SaCas9) was produced and subsequently purified via affinity chromatography. The purified apoenzyme was supplemented with ten different sgRNAs, and the nuclease activity was confirmed by the linearization of plasmid DNA containing cloned DNA target sequences.

The signaling molecular mechanisms in zebrafish response to electricity are unknown, so here we asked if changes to dopaminergic signaling pathways can affect their electrically-evoked locomotion. To answer this question, the effects of multiple selective and non-selective dopamine compounds on the electric response of zebrafish larvae is investigated. A microfluidic device with enhanced control of experimentation with multiple larvae is used, which features a novel design to immobilize four zebrafish larvae in parallel and expose them to electric current that induces tail locomotion. In 6 days post-fertilization zebrafish larvae, the electric induced locomotor response is quantified in terms of the tail movement duration and beating frequency to discern the effect of non-lethal concentrations of dopaminergic agonists (apomorphine, SKF-81297, and quinpirole), and antagonists (butaclamol, SCH-23390, and haloperidol). All dopamine antagonists decrease locomotor activity, while dopamine agonists do not induce similar behaviours in larvae. The D2- like selective dopamine agonist quinpirole enhances movement. However, exposure to non-selective and D1-selective dopamine agonists apomorphine and SKF-81297 cause no significant change in the electric response. Exposing larvae that were pre-treated with butaclamol and haloperidol to apomorphine and quinpirole, respectively, restores electric locomotion. The results demonstrate a correlation between electric response and the dopamine signalling pathway. We propose that the electrofluidic assay has profound application potential as a chemical screening method when investigating biological pathways, behaviors, and brain disorders.

The chloroplast represents an attractive compartment for light-driven biosynthesis of recombinant products, and advanced synthetic biology tools are available for engineering the chloroplast genome (=plastome) of several algal and plant species. However, producing commercial lines will likely require several plastome manipulations, and this will present issues with respect to selectable markers: there are a limited number of markers available, these can be used only once in a serial engineering strategy, and it is undesirable to retain marker genes for antibiotic resistance in the final transplastome. To address these problems, we have designed a rapid iterative marker system for the green microalga Chlamydomonas reinhardtii that allows creation of marker-free transformants starting from wild-type strains. The system employs a dual marker encoding a fusion protein of E. coli aminoglycoside adenyltransferase (conferring spectinomycin resistance) and a variant of E. coli cytosine deaminase (conferring sensitivity to 5-fluorocytosine). Initial selection on spectinomycin allows stable transformants to be established and driven to homoplasmy. Subsequent selection on 5-fluorocytosine results in rapid loss of the dual marker through intramolecular recombination between the marker’s 3’UTR and the 3’UTR of the introduced transgene(s). We demonstrate the versatility of the CpPosNeg system by serial introduction of reporter genes into the plastome.